U.S. patent application number 12/823098 was filed with the patent office on 2011-04-07 for recombinant elastase proteins and methods of manufacturing and use thereof.
Invention is credited to KIMBERLY S. BLAND, BEE C. DING, F. NICHOLAS FRANANO, MARCO D. WONG.
Application Number | 20110081705 12/823098 |
Document ID | / |
Family ID | 40344763 |
Filed Date | 2011-04-07 |
United States Patent
Application |
20110081705 |
Kind Code |
A1 |
FRANANO; F. NICHOLAS ; et
al. |
April 7, 2011 |
RECOMBINANT ELASTASE PROTEINS AND METHODS OF MANUFACTURING AND USE
THEREOF
Abstract
The present invention relates to methods for the manufacture,
purification, formulation, and use of biologically active
recombinant elastase proteins. Described are recombinant methods
for producing therapeutically useful elastase proteins, as are
pharmaceutical compositions comprising said elastase proteins.
Novel recombinant elastase proteins and protein preparations are
also disclosed. Methods are described for treating and preventing
diseases of biological conduits using pharmaceutical compositions
containing the elastase proteins of the invention.
Inventors: |
FRANANO; F. NICHOLAS;
(KANSAS CITY, MO) ; BLAND; KIMBERLY S.; (KANSAS
CITY, MO) ; WONG; MARCO D.; (OVERLAND PARK, KS)
; DING; BEE C.; (OVERLAND PARK, KS) |
Family ID: |
40344763 |
Appl. No.: |
12/823098 |
Filed: |
June 24, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12746509 |
Sep 21, 2010 |
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PCT/US2008/085559 |
Dec 4, 2008 |
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12823098 |
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60992319 |
Dec 4, 2007 |
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Current U.S.
Class: |
435/218 ;
435/243; 435/320.1; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
C12N 15/815 20130101;
C07K 2319/00 20130101; A61P 9/00 20180101; C12N 9/64 20130101; A61K
38/486 20130101; C12N 9/6448 20130101; A61P 43/00 20180101; C07K
2319/50 20130101; C12P 21/02 20130101; A61P 9/10 20180101; C07K
1/14 20130101; C12Y 304/21036 20130101; C12Y 304/21037 20130101;
A61K 9/19 20130101; A61K 38/00 20130101; C07K 2319/02 20130101;
C12N 9/50 20130101 |
Class at
Publication: |
435/218 ;
530/350; 536/23.5; 435/320.1; 435/243; 435/69.1 |
International
Class: |
C12N 9/66 20060101
C12N009/66; C07K 14/00 20060101 C07K014/00; C07H 21/00 20060101
C07H021/00; C12N 15/63 20060101 C12N015/63; C12N 1/15 20060101
C12N001/15; C12P 21/00 20060101 C12P021/00 |
Claims
1. A proelastase protein comprising (i) optionally, a signal
sequence; (ii) an elastase activation peptide sequence comprising
an elastase recognition sequence operably linked to (iii) an amino
acid sequence of a type I mature elastase.
2. The proelastase protein of claim 1, wherein said elastase
recognition sequence comprises the amino acid sequence of SEQ ID
NO:119.
3. The proelastase protein of claim 2, wherein proline is at the P2
position of said elastase recognition sequence.
4. The proelastase protein of claim 1, wherein said elastase
recognition sequence comprises the amino acid sequence of SEQ ID
NO:124.
5. The proelastase protein of claim 4, wherein proline is at the P2
position of said elastase recognition sequence.
6. The proelastase protein of claim 1 which comprises a cleavage
domain the propeptide portion of which comprises the amino acid
sequence of SEQ ID NO:120.
7. The proelastase protein of claim 4, wherein proline is at the P2
position of said cleavage domain.
8. The proelastase protein of claim 4, wherein histidine is at the
P5 position of said cleavage domain.
9. The proelastase protein of claim 1 which comprises a cleavage
domain of SEQ ID NO:123 or SEQ ID NO:125.
10. The proelastase protein of claim 1, wherein said activation
peptide sequence comprises the amino acid sequence of SEQ ID
NO:121.
11. The proelastase protein of claim 1, wherein the amino acid
sequence of said type I mature elastase has at least 90% sequence
identity to the amino acid sequence from position 6 to the end of
SEQ ID NO:84 or SEQ ID NO:1.
12. The proelastase protein of claim 1 which comprises a signal
sequence.
13. The proelastase protein of claim 1 which is isolated.
14. A nucleic acid encoding a proelastase protein of claim 1.
15. A vector comprising the nucleic acid molecule of claim 14.
16. A host cell genetically engineered to express the nucleic acid
molecule of claim 14.
17. A solution comprising the proelastase protein of any one of
claim 1.
18. The solution of claim 17 which is a buffer solution.
19. The solution of claim 18, wherein the buffer solution comprises
phosphate.
20. The solution of claim 18, wherein the buffer solution comprises
Tris base.
21. A cell culture supernatant comprising the proelastase protein
of claim 1.
22. A method of producing a proelastase protein, comprising
culturing the host cell of claim 16 under conditions in which the
proelastase protein is produced.
23. The method of claim 22, further comprising recovering the
proelastase protein.
24. A method of producing a mature elastase protein, comprising
subjecting the solution of claim 17 to conditions that produce
mature elastase protein.
25. The method of claim 24, wherein said conditions do not include
contacting said proelastase protein with trypsin.
26. The method of claim 24, wherein said conditions include
contacting said proelastase protein with a catalytic amount of an
elastase.
27. The method of claim 24, wherein said conditions include
subjecting the solution to a pH of 6 to 11.
28. The method of claim 24, wherein said solution comprises cell
culture components.
29. A method of producing a maturing elastase protein, comprising:
(a) culturing the host cell of claim 16 under conditions in which
the proelastase protein is produced; (b) recovering the proelastase
protein, and (c) subjecting a solution comprising said proelastase
protein to conditions that produce a mature elastase protein,
thereby producing a mature elastase protein.
30. The method of claim 29, further comprising the step of
lyophilizing the mature elastase protein.
31. The method of claim 29, further comprising the step of
purifying the proelastase protein.
32. The method of claim 29, further comprising the step of
purifying the mature elastase protein.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 12/746,509, U.S. national stage of
international application no. PCT/US2008/085559, filed Dec. 4,
2008, which claims the priority benefit of U.S. application Ser.
No. 12/327,809, filed Dec. 3, 2008 and of U.S. provisional
application No. 60/992,319, filed Dec. 4, 2007, the contents of
each of which are incorporated by reference herein in their
entireties.
1. FIELD OF THE INVENTION
[0002] The present invention relates to recombinant methods of
manufacturing and formulating elastase proteins for use in treating
and preventing diseases of biological conduits. The present
invention further relates to novel recombinant elastase proteins
and pharmaceutical compositions containing such proteins. Yet
further, the present invention relates to the use of pharmaceutical
compositions comprising recombinant elastase proteins for the
treatment and prevention of diseases of biological conduits.
2. BACKGROUND OF THE INVENTION
[0003] Elastin is a protein capable of spontaneously recoiling
after being stretched. Cross-linked elastin is the major structural
component of elastic fibers, which confers tissue elasticity. A
proteinase may be named an elastase if it possesses the ability to
solubilize mature, cross-linked elastin (Bieth, J G "Elastases:
catalytic and biological properties," at pp. 217-320 (Mecham
Edition, Regulation of Matrix Accumulation, New York, Academic
Press, 1986). Several published patent applications (WO 2001/21574;
WO 2004/073504; and WO 2006/036804) indicate that elastase, alone
and in combination with other agents, is beneficial in the
treatment of diseases of biological conduits, including biological
conduits which are experiencing, or susceptible to experiencing,
obstruction and vasospasm. For elastase therapy of human subjects,
the use of a human elastase is desirable to reduce the risk of
immune reaction to a non-human elastase.
[0004] To this date, however, there is no known commercially viable
means of producing biologically active elastase in sufficiently
pure form and in sufficient quantities for clinical applications.
Because elastases are powerful proteases that can hydrolyze
numerous proteins other than elastin, the proteolytic activity of
elastase poses potential obstacles for its recombinant production.
For example, the activity of mature elastase can damage the host
cell which is expressing it, degrade itself, or degrade agents used
to assist in the production or characterization of the
elastase.
[0005] Elastases are often expressed as preproproteins, containing
a signal peptide, an activation peptide, and a mature, active
portion. Cleavage of the signal sequence upon secretion yields a
proprotein that can have little or no enzymatic activity, and whose
amino acid sequence contains the amino acid sequence of an
activation peptide and a mature protein. Generally, for recombinant
expression, an inactive precursor may be expressed instead of the
mature active enzyme to circumvent damage to the cell that
expresses it. For example, U.S. Pat. No. 5,212,068 describes the
cloning of human pancreatic elastase cDNAs (referred to therein as
elastase "IIA," "IIB," "IIIA" and "IIIB"). The various elastases
were expressed as full-length cDNAs, including the native signal
sequences, in mammalian COS-1 cells. In addition, engineered
versions of the elastases, containing a B. subtilis signal sequence
and a .beta.-galactosidase signal sequence, were also expressed in
B. subtilis and E. coli, respectively. U.S. Pat. No. 5,212,068 also
suggests expressing elastases in S. cerevisiae. Generally, working
examples of elastase expression in U.S. Pat. No. 5,212,068 show low
activity of the recovered elastase or require an activation step
involving treatment with trypsin, to generate the active elastase.
In addition, the elastases were largely present in inclusion bodies
when expressed in E. coli, and only small portions of the expressed
elastase were soluble and active. None of the elastase preparations
described in U.S. Pat. No. 5,212,068 was purified to pharmaceutical
grade.
[0006] Thus, there is a need in the art for recombinant
manufacturing methods that allow the generation of therapeutic
amounts of biologically active pharmaceutical grade elastases, and
preferably avoid a trypsin activation step that is costly for
large-scale preparation and can result in trypsin contamination of
the final product. Administration of an elastase containing trypsin
to a patient could result in activation of the protease-activated
receptors 1 and 2, which may reduce some of the beneficial effects
of elastase treatment.
[0007] Citation or identification of any reference in Section 2 or
in any other section of this application shall not be construed as
an admission that such reference is available as prior art to the
present invention.
3. SUMMARY OF THE INVENTION
[0008] The present invention provides novel, efficient methods of
making recombinant elastase proteins and the use of the recombinant
proteins in compositions, e.g., pharmaceutical compositions,
elastase formulations or unit dosages, for the treatment and
prevention of diseases of biological conduits.
[0009] The present invention is directed to auto-activated
proelastase proteins, nucleic acids encoding auto-activated
proelastase proteins, host cells comprising said nucleic acids,
methods of making auto-activated proelastase proteins and the use
of auto-activated proelastase proteins in the manufacture of
mature, biologically active elastase proteins, for example for use
in pharmaceutical formulations. The term "auto-activated" (or
"autoactivated") is used herein interchangeably with the terms
"auto-activating," "self-activating," and "self-activated" and is
not intended to imply that an activation step has taken place. The
term "activation" is used herein interchangeably with the term
"conversion" and is not intended to imply that a protein resulting
from "activation" necessarily possesses enzymatic activity.
[0010] As used hereinbelow, and unless indicated otherwise, the
term "elastase" generally refers to mature elastase proteins with
elastase activity as well as immature elastase proteins, including
immature proelastase proteins (also referred to herein as elastase
proproteins) and immature preproelastase proteins (also referred to
herein as elastase preproproteins).
[0011] Preferred elastases of the invention are type I pancreatic
elastases, e.g., human type I pancreatic elastase and porcine type
I pancreatic elastase. Type I pancreatic elastases are sometimes
referred to herein as "elastase-1," "elastase 1," "elastase type
1," "type 1 elastase" or "ELA-1." Human type I pancreatic elastase
is also referred to herein as hELA-1 or human ELA-1, and porcine
type I pancreatic elastase is also referred to herein as pELA-1 or
porcine ELA-1.
[0012] A mature elastase protein of the invention typically has an
amino acid sequence encoded by a naturally occurring elastase gene
or a variant of such sequence. Preferred sequence variants,
including variants containing amino acid substitutions, are
described herein. A proelastase protein is a largely inactive
precursor of a mature elastase protein, and a preproelastase
protein further contains a signal sequence for protein secretion.
Pre and pro sequences of the elastase proteins of the invention are
typically not native to the elastase genes encoding the mature
elastase proteins of the invention. Thus, in a sense, the immature
elastase proteins of the invention are "chimeric" proteins, with
their mature portions encoded by a naturally-occurring elastase
gene and their immature portions encoded by non-elastase gene
sequences.
[0013] For ease of reference, the elastase proteins of the
invention and their core sequence components are depicted in FIG.
2. As shown in FIG. 2, the amino acid residues within the
proelastase sequence that are N-terminal to the cleavage bond
(i.e., the bond that is cleaved to yield mature elastase protein)
are designated herein as PX, . . . P5, P4, P3, P2, and P1, where P1
is immediately N-terminal to the cleavage bond, whereas amino acids
residues located to the C-terminus to the cleavage bond (and to the
N-terminus) of the mature elastase protein are designated P1', P2',
P3', . . . PX', where P1' is immediately C-terminal to the cleavage
bond and represents the N-terminal amino acid residue of the mature
protein. FIG. 2 also shows the following components:
[0014] (1) SIGNAL SEQUENCE: A sequence that increases the
proportion of expressed molecules that are secreted from the cell.
An exemplary sequence is amino acids 1-22 of SEQ ID NOS:50 or
51.
[0015] (2) PROPEPTIDE+SPACER: An optional, preferably a
non-elastase, propeptide sequence (such as yeast .alpha.-factor
propropeptide) that can further optionally include one or more
spacer sequences (a yeast .alpha.-factor propeptide sequences and
Kex2 and STE13 spacer sequences are depicted in FIG. 1B). In a
specific embodiment, the propeptide sequence does not include a
spacer.
[0016] (3) ELASTASE PROPEPTIDE: Peptide that, when present on the
N-terminal end of an elastase, renders the molecule inactive or
less active as compared to the corresponding mature elastase
protein. The elastase propeptide may be contiguous with the
activation peptide or may contain additional amino acids relative
to the activation peptide. Exemplary elastase propeptide sequences
are amino acids 1-10 of SEQ ID NOS:64 and 69.
[0017] (4) ACTIVATION PEPTIDE: Used interchangeably herein with
"activation sequence," an activation peptide is a component of, and
can be contiguous with, the elastase propeptide. As shown in FIG.
2, the activation peptide contains amino acid residues P10 through
P1. An exemplary activation peptide consensus sequence is SEQ ID
NO:80 or SEQ ID NO:121; other examples of activation peptide
sequences are SEQ ID NOS: 23, 72 and 73.
[0018] (5) RECOGNITION SEQUENCE: A recognition sequence is a
component of the elastase propeptide. As shown in FIG. 2, the
recognition sequence contains amino acid residues P3 through P1.
Exemplary recognition sequence consensus sequences are SEQ ID
NOS:11-13, 93, and 119, and an exemplary recognition sequence is
SEQ ID NO:20.
[0019] (6) CLEAVAGE DOMAIN: A region in the proelastase protein
that spans the cleavage bond. As shown in FIG. 2, the cleavage
domain contains amino acid residues P5 through P3'. Exemplary
cleavage domain sequences are SEQ ID NOS:42, 43, 48, 49, 53, 53, 54
and 55.
[0020] (7) CLEAVAGE SITE: Another region in the proelastase protein
that also spans the cleavage bond. As shown in FIG. 2, the cleavage
site contains amino acid residues P4 through P4'. An exemplary
cleavage site sequence is SEQ ID NO:27.
[0021] (8) PREPROELASTASE PROTEIN: A protein that can comprise all
of the component parts. An exemplary preproelastase protein can
comprise a peptides of SEQ ID NO:50, 51, 96, or 97 followed by an
operably linked protein of SEQ ID NO:64 or SEQ ID NO:69.
[0022] (9) PROELASTASE PROTEIN: A protein that comprises mature
elastase protein, an elastase propeptide, and the optional
propeptide and spacer sequences. Exemplary proelastase sequences
are SEQ ID NOS:64 and 69.
[0023] (10) MATURE ELASTASE PROTEIN: A protein that when properly
processed displays elastase activity. Exemplary mature sequences
are SEQ ID NO:1 (human) and SEQ ID NO:39 (porcine).
[0024] The elastase protein components can be considered modular
building blocks of the elastase proteins, proelastase proteins and
preproelastase proteins. For example, the present invention
provides a proelastase protein comprising the sequence of an
elastase propeptide and a mature elastase protein. The elastase
propeptide can contain an activation peptide. The elastase
propeptide can also contain an elastase recognition sequence. The
present invention also provides a proelastase protein comprising a
cleavage domain or cleavage site in the region spanning the
junction between the elastase propeptide and the mature elastase
protein. The proelastase proteins may further comprise a signal
sequence for secretion. Such proteins are considered preproelastase
proteins. The preproelastase proteins may further comprise a yeast
alpha factor propeptide and optionally a spacer sequence between
the signal sequence and the elastase propeptide. The elastase
proteins of the invention may also contain components in addition
to the core modular components illustrated in FIG. 2. For example,
an elastase protein can contain an epitope tag or a histidine tag
for purification. It should also be noted that the elastase
proteins of the invention need not contain all the components
depicted in FIG. 2, but generally contain at least one of the
components (including, by way of example but not limitation, a
mature elastase or a proelastase sequence) depicted in FIG. 2.
Exemplary elastase proteins of the invention are set forth in
embodiments 1-12, 28-39 and 68-69 in Section 8 below, including
exemplary type I proelastase proteins set forth in embodiments
13-27 in Section 8 below.
[0025] In certain aspects, the proelastase proteins of the
disclosure have an elastase recognition sequence P3-P2-P1, wherein
the amino acid residue at the P3 position is selected from: [0026]
(a) any of the 26 natural amino acid residues except proline or
glycine; [0027] (b) alanine, leucine, isoleucine, methionine,
lysine, asparagine, histidine, threonine, or valine; and [0028] (c)
alanine, leucine, isoleucine, methionine, lysine, asparagine, or
valine;
[0029] wherein the amino acid residue at the P2 position
independently selected from: [0030] (a) proline, alanine, leucine,
isoleucine, glycine, valine, histidine asparagine, or threonine;
[0031] (b) proline, alanine, leucine, isoleucine, glycine, valine,
asparagine, threonine; [0032] (c) proline, alanine, leucine,
isoleucine, glycine, valine, threonine; and [0033] (d) proline;
[0034] and wherein the amino acid residue at the P1 position is
independently selected from: [0035] (a) alanine, leucine, valine,
isoleucine or serine; [0036] (b) alanine, leucine, valine,
isoleucine, asparagine or serine; and [0037] (c) alanine
[0038] In other aspects, the proelastase proteins of the disclosure
have a propeptide portion of an elastase cleavage domain
P5-P4-P3-P2-P1, wherein the amino acid residue at the P5 position
is selected from: [0039] (a) any of the 26 natural amino acid
residues; [0040] (b) glutamate, histidine, proline, glycine,
asparagine, lysine or alanine, [0041] (c) histidine or glutamate;
and [0042] (d) histidine,
[0043] wherein the amino acid at the P4 position is independently
selected from: [0044] (a) any of the 26 natural amino acid residues
except glycine, lysine, phenylalanine, tyrosine, tryptophan, or
arginine; and [0045] (b) threonine, alanine, proline, or
histidine,
[0046] wherein the amino acid residue at the P3 position is
independently selected from: [0047] (a) any of the 26 natural amino
acid residues except proline or glycine; [0048] (b) alanine,
leucine, isoleucine, methionine, lysine, asparagine, histidine,
threonine, or valine; and [0049] (c) alanine, leucine, isoleucine,
methionine, lysine, asparagine, or valine;
[0050] wherein the amino acid residue at the P2 position is
independently selected from: [0051] (a) proline, alanine, leucine,
isoleucine, glycine, valine, histidine asparagine, or threonine;
[0052] (b) proline, alanine, leucine, isoleucine, glycine, valine,
asparagine, threonine; [0053] (c) proline, alanine, leucine,
isoleucine, glycine, valine, threonine; and [0054] (d) proline;
[0055] and wherein the amino acid residue at the P1 position is
independently selected from: [0056] (a) alanine, leucine, valine,
isoleucine or serine; [0057] (b) alanine, leucine, valine,
isoleucine, asparagine or serine; and [0058] (c) alanine
[0059] In certain aspects, the proelastase proteins of the
disclosure have an elastase cleavage domain in which: [0060] the
amino acid residue at the P1' position is alanine, leucine, valine,
isoleucine or serine, and is most preferably valine; [0061] the
amino acid residue at the P2' position is glycine, alanine, or
valine, and is most preferably valine; [0062] the amino acid
residue at the P3' position is glycine, valine, threonine,
phenylalanine, tyrosine or tryptophan, and is most preferably
glycine.
[0063] In other aspects, the proelastase proteins of the disclosure
have an activation peptide in which: [0064] the amino acid residue
at the P10 position is threonine; [0065] the amino acid residue at
the P9 position is glutamine or histidine; [0066] the amino acid
residue at the P8 position is aspartate; [0067] the amino acid
residue at the P7 position is leucine; and [0068] the amino acid
residue at the P6 position is proline.
[0069] In certain preferred embodiments, a proelastase protein of
the disclosure has a histidine residue at the P5 position and/or a
proline residue at the P2 position and/or an alanine residue at the
P1 position.
[0070] The present invention provides proelastase proteins
comprising (i) optionally, a signal sequence; (ii) an elastase
activation peptide sequence comprising an elastase recognition
sequence operably linked to (iii) an amino acid sequence of a type
I mature elastase.
[0071] In certain aspects, the proelastase protein has an elastase
recognition sequence of SEQ ID NO:119 or SEQ ID NO:124.
[0072] In certain aspects, the proelastase protein has a cleavage
domain the propeptide portion of which comprises the amino acid
sequence of SEQ ID NO:120.
[0073] In certain aspects, the proelastase protein has a cleavage
domain of SEQ ID NO:123 or SEQ ID NO:125.
[0074] In certain aspects, the proelastase protein has an
activation peptide sequence of SEQ ID NO:121.
[0075] In certain aspects, the proelastase proteins of the
invention are isolated.
[0076] Nucleic acids encoding the elastase proteins of the
invention, methods for producing and purifying the proteins,
recombinant cells and cell culture supernatants, compositions
comprising elastase proteins (e.g., pharmaceutical compositions,
unit dosages, formulations), the use of the proteins for
therapeutic purposes and kits comprising the proteins,
formulations, pharmaceutical compositions and unit doses are
encompassed herein.
[0077] The present invention provides nucleic acids encoding
proelastase proteins, vectors comprising such nucleic acid, and
host cells genetically engineered to express proelastase proteins,
for example a host cells into which a vector encoding a proelastase
protein is introduced.
[0078] The present invention provides a solution comprising a
proelastase protein. The solution can be a buffer solution and/or
contain cell culture components. In a specific embodiment, the
solution is a buffer solution comprising phosphate and/or Tris
base.
[0079] The present invention provides a cell culture supernatant
comprising a proelastase protein.
[0080] The invention provides methods of producing a mature
elastase protein, comprising subjecting a solution containing
proelastase protein to conditions that produce mature elastase
protein. The invention further provides methods of producing a
maturing elastase protein, comprising: (a) culturing a host cell
engineered to express a proelastase protein under conditions in
which the proelastase protein is produced; (b) recovering, and
optionally purifying, the proelastase protein, and (c) subjecting a
solution comprising said proelastase protein to conditions that
produce mature elastase protein, thereby producing a mature
elastase protein. In certain aspects, such conditions do not
include contacting the proelastase protein with trypsin and/or
contacting said proelastase protein with a catalytic amount of an
elastase and/or subjecting the solution to a pH of 6 to 11. In a
specific embodiment, the solution comprises cell culture
components, e.g., if it is a cell culture supernatant or is made
from cell culture supernatant containing proelastase protein. In
certain aspects, the methods further comprise the step of
lyophilizing the mature elastase protein and/or purifying the
mature elastase protein.
[0081] Nucleic acids encoding the elastase proteins of the
invention are exemplified in embodiments 40-67 in Section 8 below,
including vectors (see, e.g., embodiments 70-72). Also exemplified
in Section 8 are recombinant cells (see, e.g., embodiments 73-84),
cell supernatants containing elastase proteins (see, e.g.,
embodiment 88). Methods for producing elastase proteins are
exemplified in embodiments 89-224, 261-276 and 347-373 in Section
8. Methods for producing elastase formulations are exemplified in
embodiments 225-260 in Section 8. Methods of producing
pharmaceutical compositions are exemplified in embodiments 374-385
in Section 8. Pharmaceutical compositions comprising elastase
proteins are exemplified in embodiments 277-313 and 386 in Section
8, and unit dosages are exemplified in embodiments 413-420 in
Section 8. Formulations of elastase proteins are exemplified in
embodiments 324-346 in Section 8. The use of the elastase proteins
for therapeutic purposes is exemplified in embodiments 387-414 in
Section 8. Kits comprising the proteins are exemplified in Section
8 by way of embodiments 421-424.
[0082] Various aspects of the invention with respect to proelastase
proteins with SEQ ID NOS:64 and 69 are exemplified as Specific
Embodiments in Section 8 below; however, such embodiments are
applicable to other elastase protein sequences disclosed
herein.
[0083] The production methods described herein often include an
activation step, whereby the activation peptide is removed from the
proelastase sequence/separated from the mature elastase sequence,
thereby generating a mature elastase protein. The activation steps
described herein may be auto activation steps, i.e., carried out by
an elastase activity, or non-auto activation step, i.e.,
non-elastase mediated, e.g., carried out by trypsin.
[0084] In certain aspects, the present invention provides a nucleic
acid molecule comprising a nucleotide sequence which encodes an
elastase protein (including but not limited to a protein of any one
of SEQ ID NOS: 6-9, 64-69, 88-91, or 98-103) comprising (i) an
elastase propeptide comprising an activation peptide sequence
comprising an elastase recognition sequence operably linked to (ii)
the amino acid sequence of a protein having elastase activity. The
protein optionally further comprises a signal sequence, such as a
yeast .alpha.-factor signal peptide and exemplified by the amino
acid sequence of SEQ ID NO:34, operably linked to said elastase
propeptide. The .alpha.-factor is sometimes referred to herein as
"alph.alpha.-factor" or "alpha mating factor" or ".alpha.-mating
factor." In certain specific embodiments, the protein comprises a
non-elastase propeptide such as yeast .alpha.-factor propeptide. In
certain specific embodiments, the protein can comprise one or more
spacer sequences. Spacer sequences can include, but are not limited
to, Kex2 and STE13 protease cleavage sites. In a specific
embodiment, a Kex2 spacer can be used. In another embodiment, a
Kex2 spacer can be operably linked to STE13 spacers as shown in
FIG. 1B. A signal peptide sequence and a non-elastase propeptide
sequence is exemplified by the amino acid sequences of SEQ IDS
NO:51 or 97. A peptide containing a signal peptide sequence, a
non-elastase propeptide sequence, and a spacer sequence is
exemplified by the amino acid sequences of SEQ IDS NO:50 or 96.
[0085] In other specific embodiments, the signal sequence is a
mammalian secretion signal sequence, such as a porcine or human
type I elastase (used interchangeably with a type I pancreatic
elastase) signal sequence.
[0086] Preferably, the elastase recognition sequence is a type I
pancreatic elastase recognition sequence.
[0087] In specific embodiments, the protein having type I elastase
activity is a mature human type I elastase, for example a protein
of the amino acid sequence of SEQ ID NO:1, 4, 5, 84, or 87.
[0088] The present invention also provides a nucleic acid molecule
comprising a nucleotide sequence which encodes a protein comprising
(i) a signal sequence operable in Pichia pastoris operably linked
to (ii) an activation sequence (including but not limited to an
amino acid sequence of SEQ ID NOS: 23, 72, 73, or 80) comprising a
protease recognition sequence (including but not limited to an
amino acid sequence of any of SEQ ID NOS:11-23 and 93 which in turn
is operably linked to (iii) the amino acid sequence of a mature
human type I elastase. In a preferred embodiment, the protease
recognition sequence is a human type I elastase recognition
sequence, most preferably an elastase recognition sequence of SEQ
ID NO:20.
[0089] Proelastase proteins (optionally comprising a signal
sequence and thus representing preproelastase proteins) and mature
elastase proteins encoded by the nucleic acids of the invention are
also provided, as are compositions (e.g., pharmaceutical
compositions, formulations and unit dosages) comprising said mature
elastase proteins.
[0090] In a preferred embodiment, the proelastase protein or mature
elastase protein does not have an N-terminal methionine residue. In
another embodiment, however, the proelastase protein or mature
elastase protein does have an N-terminal methionine residue.
[0091] Table 1 below summarizes the sequence identifiers used in
the present specification. Preferred proteins of the invention
comprise or consist of any of SEQ ID NOS:1-32, 34, 37-73, 77, 78,
82-92, and 98-105 listed in Table 1 below, or are encoded in part
or entirely by the nucleotide sequences of SEQ ID NO:33 and SEQ ID
NO:81.
TABLE-US-00001 TABLE 1 Summary of amino acid and nucleotide SEQ ID
NOS. NUCLEOTIDE OR AMINO SEQ MOLECULE ACID ID NO Mature human
elastase I, including first "valine," with Amino Acid 1 possible
polymorphism at position 220 (V or L) (numbering refers to position
in context of preproprotein) Mature human elastase I, minus first
"valine," with possible Amino Acid 2 polymorphism at position 220
(V or L) (numbering refers to position in context of preproprotein)
Mature human elastase I, minus first two "valines," with Amino Acid
3 possible polymorphism at position 220 (V or L) (numbering refers
to position in context of preproprotein) Mature human elastase I,
with first "valine" substituted by Amino Acid 4 "alanine," with
possible polymorphism at position 220 (V or L) (numbering refers to
position in context of preproprotein) Mature human elastase I
(isotype 2), including first "valine" Amino Acid 5 Engineered
elastase proprotein no. 1 (pPROT42 variant), Amino Acid 6 with
possible polymorphism at position 220 (V or L) (numbering refers to
position in context of preproprotein) Engineered elastase
proprotein no. 2, with possible Amino Acid 7 polymorphism at
position 220 (V or L) (numbering refers to position in context of
preproprotein) Engineered elastase proprotein no. 3, with possible
Amino Acid 8 polymorphism at position 220 (V or L) (numbering
refers to position in context of preproprotein) Engineered elastase
proprotein no. 4, with possible Amino Acid 9 polymorphism at
position 220 (V or L) (numbering refers to position in context of
preproprotein) Engineered elastase proprotein no. 5 (pPROT24
trypsin Amino Acid 10 activated sequence), with possible
polymorphism at position 220 (V or L) (numbering refers to position
in context of preproprotein) Consensus elastase recognition
sequence 1 (Positions Amino Acid 11 Xaa.sub.1 = P3, Xaa.sub.2 = P2,
Xaa.sub.3 = P1) Consensus elastase recognition sequence 2
(Positions P3- Amino Acid 12 P2-P1) Consensus elastase recognition
sequence 3 (Positions P3- Amino Acid 13 P2-P1) Elastase recognition
sequence 1 (Positions P3-P2-P1) Amino Acid 14 Elastase recognition
sequence 2 (Positions P3-P2-P1) Amino Acid 15 Elastase recognition
sequence 3 (Positions P3-P2-P1) Amino Acid 16 Wild-type trypsin
recognition sequence (pPROT24) Amino Acid 17 (Positions P3-P2-P1)
Elastase recognition sequence 5 (Positions P3-P2-P1) Amino Acid 18
Elastase recognition sequence 6 (Positions P3-P2-P1) Amino Acid 19
Elastase recognition sequence 7 (Positions P3-P2-P1 of Amino Acid
20 Variants 48 and 55) Elastase recognition sequence 8 Amino Acid
21 Human elastase activation sequence 1 Amino Acid 22 Human
elastase activation sequence 2 Amino Acid 23 pro-PROT-201 cleavage
site Amino Acid 24 pPROT40 cleavage site Amino Acid 25 pPROT41
cleavage site Amino Acid 26 pPROT42 cleavage site Amino Acid 27
pPROT43 cleavage site Amino Acid 28 pPROT44 cleavage site Amino
Acid 29 pPROT45 cleavage site Amino Acid 30 pPROT46 cleavage site
Amino Acid 31 pPROT47 cleavage site Amino Acid 32 Coding region of
a human elastase-1 (i.e., human type I Nucleotide 33 pancreatic
elastase) (NCBI Accession No. NM_001971) Yeast alpha factor signal
peptide Amino Acid 34 20F primer Nucleotide 35 24R primer
Nucleotide 36 pPROT42 P3 Cleavage Site Variant Elastase, with
possible Amino Acid 37 polymorphism at position 220 (V or L)
(numbering refers to position in context of preproprotein) pPROT42
P2 Cleavage Site Variant Elastase, with possible Amino Acid 38
polymorphism at position 220 (V or L) (numbering refers to position
in context of preproprotein) Mature porcine pancreatic Elastase I
(from GenBank Amino Acid 39 Accession P00772.1) Elastase Variant
Propeptide Cleavage Domain 40 Amino Acid 40 Elastase Variant
Propeptide Cleavage Domain 41 Amino Acid 41 Elastase Variant
Propeptide Cleavage Domain 42 Amino Acid 42 Elastase Variant
Propeptide Cleavage Domain 43 Amino Acid 43 Elastase Variant
Propeptide Cleavage Domain 44 Amino Acid 44 Elastase Variant
Propeptide Cleavage Domain 45 Amino Acid 45 Elastase Variant
Propeptide Cleavage Domain 46 Amino Acid 46 Elastase Variant
Propeptide Cleavage Domain 47 Amino Acid 47 Elastase Variant
Propeptide Cleavage Domain 48 Amino Acid 48 Elastase Variant
Propeptide Cleavage Domain 49 Amino Acid 49 Yeast alpha-mating
factor signal peptide, propeptide, and Amino Acid 50 spacer
sequence 1 Yeast alpha-mating factor signal peptide and propeptide
Amino Acid 51 sequence 2 Elastase Variant Propeptide Cleavage
Domain 52 Amino Acid 52 Elastase Variant Propeptide Cleavage Domain
53 Amino Acid 53 Elastase Variant Propeptide Cleavage Domain 54
Amino Acid 54 Elastase Variant Propeptide Cleavage Domain 55 Amino
Acid 55 Elastase Variant Propeptide Cleavage Domain 56 Amino Acid
56 Elastase Variant Propeptide Cleavage Domain 57 Amino Acid 57
Elastase Variant Propeptide Cleavage Domain 58 Amino Acid 58
Elastase Variant Propeptide Cleavage Domain 59 Amino Acid 59
Elastase Variant Propeptide Cleavage Domain 60 Amino Acid 60
Elastase Variant Propeptide Cleavage Domain 61 Amino Acid 61
Elastase Variant Propeptide Cleavage Domain 62 Amino Acid 62
Elastase Variant Propeptide Cleavage Domain 63 Amino Acid 63
Elastase Proenzyme with variant Cleavage Domain 48, with Amino Acid
64 possible polymorphism at position 220 (V or L) (numbering refers
to position in context of preproprotein) Elastase Proenzyme with
variant Cleavage Domain 49, with Amino Acid 65 possible
polymorphism at position 220 (V or L) (numbering refers to position
in context of preproprotein) Elastase Proenzyme with variant
Cleavage Domain 52, with Amino Acid 66 possible polymorphism at
position 220 (V or L) (numbering refers to position in context of
preproprotein) Elastase Proenzyme with variant Cleavage Domain 53,
with Amino Acid 67 possible polymorphism at position 220 (V or L)
(numbering refers to position in context of preproprotein) Elastase
Proenzyme with variant Cleavage Domain 54, with Amino Acid 68
possible polymorphism at position 220 (V or L) (numbering refers to
position in context of preproprotein) Elastase Proenzyme with
variant Cleavage Domain 55, with Amino Acid 69 possible
polymorphism at position 220 (V or L) (numbering refers to position
in context of preproprotein) Wild Type Elastase + AlaArg Cleavage
Variant, with Amino Acid 70 possible polymorphism at position 220
(V or L) (numbering refers to position in context of preproprotein)
Wild Type Elastase + Arg Cleavage Variant, with possible Amino Acid
71 polymorphism at position 220 (V or L) (numbering refers to
position in context of preproprotein) Variant 48 Human Elastase
Activation peptide Amino Acid 72 Variant 55 Human Elastase
Activation peptide Amino Acid 73 Human Elastase Cleavage Domain
Consensus Sequence; Amino Acid 74 corresponds to residues P5, P4,
P3, P2, P1, P'1, P'2, and P'3 of an elastase cleavage domain PCR
Mutagenesis Primer for pPROT3 construction Nucleic Acid 75 PCR
Mutagenesis Primer or pPROT3 construction Nucleic Acid 76 Mature
ELA1 C-terminal Variant of Talas et al., 2000, Amino Acid 77 Invest
Dermatol. 114(1):165-70. Mature ELA-1 variants, with possible
polymorphisms at Amino Acid 78 positions 44 (W or R); 59 (M or V);
220 (V or L); and 243 (Q or R) (numbering refers to position in
context of preproprotein) Activation peptide variants (wild type,
trypsin cleavable), Amino Acid 79 with possible polymorphisms at
position 10 (Q or H) (numbering refers to position in context of
preproprotein) Activation peptide "consensus" sequence Amino Acid
80 Coding region of ELA-1.2A Amino Acid 81 Translation Product of
ELA-1.2A Amino Acid 82 (Trypsin activated pPROT24 sequence)
Translation Product of ELA-1.2A Amino Acid 83 (trypsin activated
pPROT24 sequence), with possible polymorphisms at positions 44 (W
or R); 59 (M or V); 220 (V or L); and 243 (Q or R) (numbering
refers to position in context of preproprotein) Mature human
elastase I, including first "valine," with Amino Acid 84 possible
polymorphisms at positions 44 (W or R); 59 (M or V); 220 (V or L);
and 243 (Q or R) (numbering refers to position in context of
preproprotein) mature human elastase I, minus first "valine," with
possible Amino Acid 85 polymorphisms at positions 44 (W or R); 59
(M or V); 220 (V or L); and 243 (Q or R) (numbering refers to
position in context of preproprotein) Mature human elastase I,
minus first two "valines," with Amino Acid 86 possible
polymorphisms at positions 44 (W or R); 59 (M or V); 220 (V or L);
and 243 (Q or R) (numbering refers to position in context of
preproprotein) mature human elastase I, with first "valine"
substituted by Amino Acid 87 "alanine," with possible polymorphisms
at positions 44 (W or R); 59 (M or V); 220 (V or L); and 243 (Q or
R) (numbering refers to position in context of preproprotein)
Engineered elastase proprotein no. 1 (pPROT42 variant), Amino Acid
88 with possible polymorphisms at positions 10 (Q or H); 44 (W or
R); 59 (M or V); 220 (V or L); and 243 (Q or R) (numbering refers
to position in context of preproprotein) Engineered elastase
proprotein no. 2, with possible Amino Acid 89 polymorphisms at
positions 10 (Q or H); 44 (W or R); 59 (M or V); 220 (V or L); and
243 (Q or R) (numbering refers to position in context of
preproprotein) Engineered elastase proprotein no. 3, with possible
Amino Acid 90 polymorphisms at positions 10 (Q or H); 44 (W or R);
59 (M or V); 220 (V or L); and 243 (Q or R) (numbering refers to
position in context of preproprotein) engineered elastase
proprotein no. 4, with possible Amino Acid 91 polymorphisms at
positions 10 (Q or H); 44 (W or R); 59 (M or V); 220 (V or L); and
243 (Q or R) (numbering refers to position in context of
preproprotein) engineered elastase proprotein no. 5 (pPROT24
trypsin Amino Acid 92 activated sequence), with possible
polymorphisms at positions 10 (Q or H); 44 (W or R); 59 (M or V);
220 (V or L); and 243 (Q or R) (numbering refers to position in
context of preproprotein) Consensus elastase recognition sequence 4
(Positions P3- Amino Acid 93 P2-P1) pPROT42 P3 Cleavage Site
Variant Elastase, with possible Amino Acid 94 polymorphisms at
positions 44 (W or R); 59 (M or V); 220 (V or L); and 243 (Q or R)
(numbering refers to position in context of preproprotein) pPROT42
P2 Cleavage Site Variant Elastase, with possible Amino Acid 95
polymorphisms at positions 44 (W or R); 59 (M or V); 220 (V or L);
and 243 (Q or R) (numbering refers to position in context of
preproprotein) Yeast alpha-mating factor signal peptide,
propeptide, and Amino Acid 96 spacer sequence 1 Yeast alpha-mating
factor signal peptide and propeptide Amino Acid 97 sequence 2
Elastase Proenzyme with variant Cleavage Domain 48 Amino Acid 98
Generic to SEQ ID NO: 64, with possible polymorphisms at positions
10 (Q or H); 44 (W or R); 59 (M or V); 220 (V or L); and 243 (Q or
R) (numbering refers to position in context of preproprotein)
Elastase Proenzyme with variant Cleavage Domain 49, with Amino Acid
99 possible polymorphisms at positions 10 (Q or H); 44 (W or R); 59
(M or V); 220 (V or L); and 243 (Q or R) (numbering refers to
position in context of preproprotein) Elastase Proenzyme with
variant Cleavage Domain 52, with Amino Acid 100 possible
polymorphisms at positions 10 (Q or H); 44 (W or R); 59 (M or V);
220 (V or L); and 243 (Q or R) (numbering refers to position in
context of preproprotein) Elastase Proenzyme with variant Cleavage
Domain 53, with Amino Acid 101 possible polymorphisms at positions
10 (Q or H); 44 (W or R); 59 (M or V); 220 (V or L); and 243 (Q or
R) (numbering refers to position in context of preproprotein)
Elastase Proenzyme with variant Cleavage Domain 54, with Amino Acid
102 possible polymorphisms at positions 10 (Q or H); 44 (W or R);
59 (M or V); 220 (V or L); and 243 (Q or R) (numbering refers to
position in context of preproprotein) Elastase Proenzyme with
variant Cleavage Domain 55, with Amino Acid 103 possible
polymorphisms at positions 10 (Q or H); 44 (W or R); 59 (M or V);
220 (V or L); and 243 (Q or R) (numbering refers to position in
context of preproprotein) Wild Type Elastase + AlaArg Cleavage
Variant, with Amino Acid 104 possible polymorphisms at positions 44
(W or R); 59 (M or V); 220 (V or L); and 243 (Q or R) (numbering
refers to position in context of preproprotein) Wild Type Elastase
+ Arg Cleavage Variant, with possible Amino Acid 105 polymorphisms
at positions 10 (Q or H); 44 (W or R); 59 (M or V); 220 (V or L);
and 243 (Q or R) (numbering refers to position in context of
preproprotein) Mature human elastase I cleavage variant lacking
first four Amino Acid 106 amino acids, with possible polymorphisms
at positions 10 (Q or H); 44 (W or R); 59 (M or V); 220 (V or L);
and 243 (Q or R) (numbering refers to position in context of
preproprotein) Mature human elastase I cleavage variant lacking
first six Amino Acid 107 amino acids, with possible polymorphisms
at positions 44 (W or R); 59 (M or V); 220 (V or L); and 243 (Q or
R) (numbering refers to position in context of preproprotein)
Mature human elastase I cleavage variant lacking first nine Amino
Acid 108
amino acids, with possible polymorphisms at positions 44 (W or R);
59 (M or V); 220 (V or L); and 243 (Q or R) (numbering refers to
position in context of preproprotein) Nucleic acid sequence of FIG.
1A Nucleic Acid 109 Amino acid sequence of FIG. 1A Amino Acid 110
Nucleic acid sequence of FIG. 1B Nucleic Acid 111 Amino acid
sequence of FIG. 1B Amino Acid 112 Nucleic acid sequence of FIG. 13
Nucleic Acid 113 Amino acid sequence of FIG. 14 Amino Acid 114
Cleavage domain sequence of trypsin-activated pPROT101- Amino Acid
115 24-V Cleavage domain sequence of auto-activated Amino Acid 116
pPROT101-42-V Cleavage domain sequence of auto-activated Amino Acid
117 pPROT101-49-V Cleavage domain sequence of auto-activated Amino
Acid 118 pPROT101-55L-V Consensus elastase recognition sequence 5
(Positions P3- Amino Acid 119 P2-P1) Consensus sequence 1 for the
propeptide portion of the Amino Acid 120 cleavage domain;
corresponds to residues P5, P4, P3, P2, and P1 Consensus sequence 2
for the activation peptide; Amino Acid 121 corresponds to residues
P10 through P1 Proelastase consensus sequence 1 for residues P10
through Amino Acid 122 P3' Consensus cleavage domain sequence 2;
corresponds to Amino Acid 123 residues P5, P4, P3, P2, P1, P'1,
P'2, and P'3 Consensus elastase recognition sequence 6 (Positions
P3- Amino Acid 124 P2-P1) Consensus cleavage domain sequence 3;
corresponds to Amino Acid 125 residues P5, P4, P3, P2, P1, P'1,
P'2, and P'3
[0092] There are at least 5 confirmed polymorphisms in human type I
elastase protein, at positions 10 (Q or H); 44 (W or R); 59 (M or
V); 220 (V or L); and 243 (Q or R). The full-length
(preproelastase) protein is 258 amino acids in length. The first 8
amino acids correspond to the signal or "pre" peptide sequence that
is cleaved off to generate an inactive proprotein, and a further
"pro" peptide sequence (comprising or consisting of 10 amino acids
corresponding to an activation peptide) are cleaved to generate the
active, mature protein. Thus, the polymorphism at position 10 is
present in the proprotein but not in the mature protein.
[0093] Accordingly, in Table 2 below are presented all possible
combinations of the 5 polymorphisms of human type I elastase. The
present invention provides preproelastase and proelastase proteins
(including but not limited to the variant preproelastase and
proelastase proteins described herein), such as the proteins
exemplified in embodiments 1-39 and 68-69 or the proteins obtained
or obtainable by the method of any one of embodiments 89-224,
261-276, and 347-373 in Section 8, comprising the any of the
combinations of polymorphisms set forth in Table 2 below.
TABLE-US-00002 TABLE 2 Variants of human type I immature elastase
(pre-pro) and pro elastase proteins. Position 10 44 59 220 243
Embodiment Q or H W or R M or V V or L Q or R 1. Q W M V Q 2. Q W M
V R 3. Q W M L Q 4. Q W V V Q 5. Q R M V Q 6. Q W M L R 7. Q W V L
Q 8. Q R V V Q 9. Q W V V R 10. Q R M L Q 11. Q R M V R 12. Q R V L
Q 13. Q R V V R 14. Q R M L R 15. Q W V L R 16. Q R V L R 17. H W M
V Q 18. H W M V R 19. H W M L Q 20. H W V V Q 21. H R M V Q 22. H W
M L R 23. H W V L Q 24. H R V V Q 25. H W V V R 26. H R M L Q 27. H
R M V R 28. H R V L Q 29. H R V V R 30. H R M L R 31. H W V L R 32.
H R V L R The position numbering refers to the position in the
context of the preproprotein of native human type I elastase.
[0094] Moreover, in Table 3 below are presented all possible
combinations of the 4 polymorphisms of human type I elastase that
may be present in a mature elastase protein. The present invention
provides mature elastase proteins (including but not limited to the
variant mature elastase proteins described herein), such as the
mature elastase proteins obtained or obtainable by the method of
any one of embodiments 89-224, 261-276, and 347-373 in Section 8,
comprising the any of the combinations of polymorphisms set forth
in Table 3 below.
TABLE-US-00003 TABLE 3 Variants of mature human type I elastase
proteins. The position numbering refers to the position in the
context of the preproprotein of native human type I elastase.
Position 44 59 220 243 Embodiment W or R M or V V or L Q or R 1. W
M V Q 2. W M V R 3. W M L Q 4. W V V Q 5. R M V Q 6. W M L R 7. W V
L Q 8. R V V Q 9. W V V R 10. R M L Q 11. R M V R 12. R V L Q 13. R
V V R 14. R M L R 15. W V L R 16. R V L R
[0095] The mature type I elastases of the invention can be
purified, for example for use in pharmaceutical compositions. In
specific embodiments, the elastases are at least 70%, 80%, 90%,
95%, 98% or 99% pure.
[0096] The mature type I elastases of the invention preferably have
a specific activity of greater than 1, greater than 5, greater than
10, greater than 20, greater than 25, or greater than 30 U/mg of
protein, as determined by measuring the rate of hydrolysis of the
small peptide substrate N-succinyl-Ala-Ala-Ala-pNitroanilide
(SLAP), which is catalyzed by the addition of elastase. One unit of
activity is defined as the amount of elastase that catalyzes the
hydrolysis of 1 micromole of substrate per minute at 30.degree. C.
and specific activity is defined as activity per mg of elastase
protein (U/mg). Preferably, a mature human type I elastase has a
specific activity within a range in which the lower limit is 1, 2,
3, 4, 5, 7, 10, 15 or 20 U/mg protein and in which the upper limit
is, independently, 5, 10, 15, 20, 25, 30, 35, 40 or 50 U/mg
protein. In exemplary embodiments, the specific activity is in the
range of from 1 to 40 U/mg of protein, from 1 to 5 U/mg of protein,
from 2 to 10 U/mg of protein, from 4 to 15 U/mg of protein, from 5
to 30 U/mg of protein, from 10 to 20 U/mg of protein, from 20 to 40
U/mg of protein, or any range whose upper and lower limits are
selected from any of the foregoing values. A mature porcine type I
elastase preferably has a specific activity within a range in which
the lower limit is 1, 2, 3, 4, 5, 7, 10, 15, 20, 30, 40, 50, 60, or
75 U/mg protein and in which the upper limit is, independently, 5,
10, 15, 20, 25, 30, 35, 40, 50, 60, 75, 90, 95 or 100 U/mg protein.
In exemplary embodiments, the specific activity of the porcine
elastase is in the range of from 10 to 50 U/mg of protein, from 20
to 60 U/mg of protein, from 30 to 75 U/mg of protein, from 30 to 40
U/mg of protein, from 20 to 35 U/mg of protein, from 50 to 95 U/mg
of protein, from 25 to 100 U/mg of protein, or any range whose
upper and lower limits are selected from any of the foregoing
values.
[0097] Accordingly, certain aspects of the present invention relate
to compositions, such as pharmaceutical compositions, elastase
formulations and unit dosages, such as those exemplified in
embodiments 277-314, 346, 386, and 415-420 or those obtained or
obtainable by the method of any one of embodiments 261-276 and
374-385 in Section 8 below.
[0098] In certain embodiments, the compositions of the invention
comprise trypsin-activated elastase proteins, e.g., trypsin
activated proteins made by any of the methods disclosed herein. In
other embodiments, the compositions comprise autoactivated elastase
proteins, e.g., autoactivated elastase proteins made by any of the
methods disclosed herein. In certain aspects, a composition of the
invention is characterized by at least one, at least two, at least
three, at least four, at least five, at least six or at least seven
of the following properties: (a) the composition is free of
trypsin; (b) the composition is substantially free of trypsin; (c)
the composition is free of any protein consisting of SEQ ID NOS:70
and 71; (d) the composition is substantially free of any protein
consisting of SEQ ID NOS:2 and 3; (e) the composition is free of
bacterial proteins; (f) the composition is substantially free of
bacterial proteins; (g) the composition is free of mammalian
proteins other than said mature elastase protein; (h) the
composition is substantially free of mammalian proteins other than
said mature elastase protein; (i) the composition is free or
substantially free of one, two, three or all four proteins
consisting of SEQ ID NO:85, 86, 94 and 95; (j) the composition is
free or substantially free of one, two, or all three proteins
consisting of SEQ ID NO:106, 107 and 108; (k) the composition
contains pharmaceutically acceptable levels of endotoxins (e.g., 10
EU/mg elastase or less, or 5 EU/mg elastase or less); (l) the
mature elastase protein in the composition is characterized by a
specific activity of 1 to 40 U/mg of protein or any other range of
specific activity disclosed herein; (m) the trypsin activity in
said composition corresponds to less than 4 ng per 1 mg of mature
elastase protein or any other range of trypsin activity disclosed
herein; (n) the composition comprises polysorbate-80; (o) the
composition comprises dextran; (p) the composition comprises sodium
ions, potassium ions, phosphate ions, chloride ions and
polysorbate-80; (q) the composition comprises sodium ions,
potassium ions, phosphate ions, chloride ions and dextran; (r) the
composition comprises sodium ions, potassium ions, phosphate ions,
chloride ions, polysorbate-80, and dextran; (s) the mature elastase
protein in said composition displays an amount of stability
disclosed herein, e.g., maintains 60% to 100% of its specific
activity after at least a week of storage at 4.degree. C., after at
least a month of storage at 4.degree. C., after at least two months
of storage at 4.degree. C., after at least three months of storage
at 4.degree. C., or after at least month six months of storage at
4.degree. C.; and (t) the composition comprises a unit dosage of
0.0033 mg to 200 mg of said mature elastase protein, or any other
unit dosage of mature elastase protein disclosed herein.
[0099] In certain aspects, the composition is characterized by at
least three characteristics, at least four characteristics or five
characteristics independently selected from the following groups
(i) through (v): [0100] (i) (a), (b) or (m) [0101] (ii) (e) or (f)
[0102] (iii) (g) or (h) [0103] (iv) (k) [0104] (v) (l)
[0105] In specific embodiments, two of said at least three or at
least said four characteristics are selected from groups (i) and
(iv) or (v). In other embodiments, three of at least said four
characteristics are selected from groups (i), (iv) and (v).
[0106] In specific embodiments, the present invention provides a
composition, including but not limited to a pharmaceutical
composition, elastase formulation or unit dosage, comprising (i) a
therapeutically effective amount of human type I elastase that is
free of trypsin and (ii) a pharmaceutically acceptable carrier.
Alternatively, the present invention provides a composition
comprising (i) a therapeutically effective amount of human type I
elastase that is substantially free of trypsin and (ii) a
pharmaceutically acceptable carrier. In specific embodiments, the
human type I elastase and/or the composition comprises less than 5
ng/ml of trypsin activity, less than 4 ng/ml of trypsin activity,
less than 3 ng/ml of trypsin activity, less than 2 ng/ml of trypsin
activity, or less than 1.56 ng/ml of trypsin activity. In the
foregoing examples, the ng/ml trypsin activity can be assayed in a
liquid human type I elastase composition or preparation containing
1 mg/ml human type I elastase protein. Thus, the trypsin activities
may also be described in terms of milligrams of elastase protein,
for example, less than 3 ng trypsin activity/mg elastase protein,
less than 1.56 ng trypsin activity/mg elastase protein, etc. The
present invention further provides a composition comprising (i) a
therapeutically effective amount of human type I elastase and (ii)
a pharmaceutically acceptable carrier, wherein the composition
comprises less than 5 ng of trypsin activity per mg of elastase,
less than 4 ng trypsin activity per mg of elastase, less than 3 ng
of trypsin activity per mg of elastase, less than 2 ng of trypsin
activity per mg of elastase, or less than 1.56 ng of trypsin
activity per mg of elastase.
[0107] The present invention further provides methods of improving
the quality of mature elastase proteins produced by trypsin
activation methods (e.g., the methods of embodiment 145 in Section
8 below), comprising purifying the mature elastase protein on a
Macro-Prep High S Resin column. It was found by the present
inventors that mature elastase proteins purified on a Macro-Prep
High S Resin column yields elastase compositions with trypsin
activity levels corresponding 20-25 ng trypsin activity/mg elastase
protein, as compared to purification on a Poros (Poly
(Styrene-Divinylbenzene)) column which yields elastase compositions
with trypsin activity levels corresponding to approximately 1000 ng
trypsin activity/mg elastase protein. The present invention further
provides elastase compositions comprising mature elastase proteins
produced by purifying trypsin-activated elastase proteins on a
Macro-Prep High S Resin column. The Macro-Prep High S Resin is
available from Biorad (Hercules, Calif.), according to whom a
column of rigid methacrylate supports with little shrinkage and
swelling. Other similar cation exchange columns of the same class
and/or with the same bead size (around 50 .mu.m) may be used, such
as other methacrylate columns, may suitably be used.
[0108] Other aspects of the present invention relate to
compositions, including but not limited to pharmaceutical
compositions, elastase formulations or unit dosages, comprising
porcine type I pancreatic elastase. In specific embodiments, the
present invention provides a composition comprising (i) a
therapeutically effective amount of porcine type I pancreatic
elastase that is free of trypsin and (ii) a pharmaceutically
acceptable carrier. Alternatively, the present invention provides a
composition comprising (i) a therapeutically effective amount of
porcine type I pancreatic elastase that is substantially free of
trypsin and (ii) a pharmaceutically acceptable carrier. In specific
embodiments, the porcine type I pancreatic elastase and/or the
composition comprises less than 100 ng/ml of trypsin activity, less
than 75 ng/ml of trypsin activity, less than 50 ng/ml of trypsin
activity, less than 25 ng/ml of trypsin activity, less than 15
ng/ml of trypsin activity, less than 10 ng/ml of trypsin activity,
less than 5 ng/ml of trypsin activity, less than 4 ng/ml of trypsin
activity, less than 3 ng/ml of trypsin activity, less than 2 ng/ml
of trypsin activity, or less than 1.56 ng/ml of trypsin activity.
In the foregoing examples, the ng/ml trypsin activity can be
assayed in a liquid porcine type I pancreatic elastase composition
or preparation containing 1 mg/ml porcine type I pancreatic
elastase. Thus, the trypsin activities may also be described in
terms of milligrams of elastase protein, for example, less than 25
ng trypsin activity/mg elastase protein, less than 5 ng trypsin
activity/mg elastase protein, etc. The present invention further
provides a composition comprising (i) a therapeutically effective
amount of porcine type I elastase and (ii) a pharmaceutically
acceptable carrier, wherein the composition comprises than 100 ng
of trypsin activity per mg of elastase, less than 75 ng trypsin
activity per mg of elastase, less than 50 ng of trypsin activity
per mg of elastase, less than 25 ng of trypsin activity per mg of
elastase, less than 15 ng of trypsin activity per mg of elastase,
less than 10 ng or trypsin activity per mg of elastase, less than 5
ng of trypsin activity per mg of elastase, less than 4 ng trypsin
activity per mg of elastase, less than 3 ng of trypsin activity per
mg of elastase, less than 2 ng of trypsin activity per mg of
elastase, or less than 1.56 ng of trypsin activity per mg of
elastase.
[0109] In other embodiments, the present invention provides
compositions of elastase proteins, such as mature elastase
proteins, including but not limited to pharmaceutical compositions,
elastase formulations or unit dosages, that are free of N-terminal
variants corresponding to one, two, three or all four of SEQ ID
NOS: 70, 71, 104, 105. In certain embodiments, the present
invention provides a pharmaceutical composition comprising (i) a
therapeutically effective amount of mature human type I elastase
(ii) a pharmaceutically acceptable carrier, which pharmaceutical
composition is free of any protein with SEQ ID NOS:70, 71, 104,
105.
[0110] In other embodiments, the present invention provides a
composition, including but not limited to a pharmaceutical
composition, an elastase formulation or unit dosage, comprising (i)
a therapeutically effective amount of human type I elastase that is
free or substantially free of variant proteins containing specific
additional amino acids on the N-terminal end of the mature elastase
protein (SEQ ID NOS: 37, 38, 70, 71, 94, 95, 104, 105) and (ii) a
pharmaceutically acceptable carrier. In other embodiments, the
present invention provides a composition comprising (i) a
therapeutically effective amount of human type I elastase that is
free or substantially free of variant proteins lacking N-terminal
amino acids of the mature elastase protein (SEQ ID NOS: 2, 3, 37,
38, 70, 71, 85, 86, 94, 95, 104, 105, 106, 107, 108) and (ii) a
pharmaceutically acceptable carrier. In specific embodiments, the
human type I elastase and/or the composition comprises less than
25% N-terminal variants, less than 20% N-terminal variants, less
than 15% N-terminal variants, less than 10% N-terminal variants,
less than 5% N-terminal variants, less than 4% N-terminal variants,
less than 3% N-terminal variants, less than 2% N-terminal variants,
less than 1% N-terminal variants, less than 0.5% N-terminal
variants, 0% N-terminal variants, or comprises N-terminal variants
in an amount ranging between any two of the foregoing percentages
(e.g., 2%-25% N-terminal variants, 0.5%-10% N-terminal variants,
5%-15% N-terminal variants, 0%-2% N-terminal variants, etc.). As
used herein, the term "less than X % N-terminal variants" refers to
the amount of N-terminal variants as a percentage of total elastase
protein.
[0111] In other embodiments, the present invention provides a
composition, including but not limited to a pharmaceutical
composition, an elastase formulation or unit dosage, comprising (i)
a therapeutically effective amount of mature human type I elastase
(ii) a pharmaceutically acceptable carrier, which pharmaceutical
composition is substantially free of bacterial proteins and/or is
substantially free of mammalian proteins other than said mature
human type I elastase. In specific embodiments, the human type I
elastase and/or the composition comprises less than 25% bacterial
proteins and/or mammalian proteins other than said mature human
type I elastase, less than 20% bacterial proteins and/or mammalian
proteins other than said mature human type I elastase, less than
15% bacterial proteins and/or mammalian proteins other than said
mature human type I elastase, less than 10% bacterial proteins
and/or mammalian proteins other than said mature human type I
elastase, less than 5% bacterial proteins and/or mammalian proteins
other than said mature human type I elastase, less than 4%
bacterial proteins and/or mammalian proteins other than said mature
human type I elastase, less than 3% bacterial proteins and/or
mammalian proteins other than said mature human type I elastase,
less than 2% bacterial proteins and/or mammalian proteins other
than said mature human type I elastase, less than 1% bacterial
proteins and/or mammalian proteins other than said mature human
type I elastase, less than 0.5% bacterial proteins and/or mammalian
proteins other than said mature human type I elastase, 0% bacterial
proteins and/or mammalian proteins other than said mature human
type I elastase, or comprises bacterial proteins and/or mammalian
proteins other than said mature human type I elastase in an amount
ranging between any two of the foregoing percentages (e.g., 2%-25%
bacterial proteins and/or mammalian proteins other than said mature
human type I elastase, 0.5%-10% bacterial proteins and/or mammalian
proteins other than said mature human type I elastase, 5%-15%
bacterial proteins and/or mammalian proteins other than said mature
human type I elastase, 0%-2% bacterial proteins and/or mammalian
proteins other than said mature human type I elastase, etc.). As
used herein, the term "less than X % bacterial proteins and/or
mammalian proteins other than said mature human type I elastase"
refers to the amount of such proteins as a percentage of total
protein in an elastase preparation or in said composition. In
certain embodiments, the elastase represents at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at
least 99.8% of the total protein in such compositions or
preparations.
[0112] Methods for the treatment and prevention of diseases of
biological conduits, comprising administration of compositions
(e.g., pharmaceutical compositions, elastase formulations or unit
dosages) comprising a purified mature human type I elastase of the
invention to a patient in need thereof, are also provided.
[0113] Further provided are vectors comprising nucleic acids
encoding any of the elastase proteins of the invention ("nucleic
acids of the invention"), host cells engineered to express the
nucleic acids of the invention. In specific embodiments, the
vectors further comprise a nucleotide sequence which regulates the
expression of the elastase protein. For example, the nucleotide
sequence encoding the protein of the invention can be operably
linked to a methanol-inducible promoter. Other suitable promoters
are exemplified in Section 5.3 below.
[0114] In a specific embodiment, the present invention provides a
vector comprising a nucleotide sequence encoding an open reading
frame, the open reading frame comprising a yeast .alpha.-factor
signal peptide or a type I elastase signal peptide (e.g., porcine
elastase signal peptide) operably linked to a human type I elastase
proprotein sequence. Optionally, the vector further comprises a
methanol-inducible promoter operably linked to the open reading
frame.
[0115] Host cells comprising the nucleic acids and vectors of the
invention are also provided. In certain embodiments, the vector or
nucleic acid is integrated into the host cell genome; in other
embodiments, the vector or nucleic acid is extrachromosomal. A
preferred host cell is a Pichia pastoris cell. Other suitable host
cells are exemplified in Section 5.3 below.
[0116] In a specific embodiment, the present invention provides a
Pichia pastoris host cell genetically engineered to encode an open
reading frame comprising a yeast .alpha.-factor signal peptide or a
porcine elastase signal peptide operably linked to a human type I
elastase proenzyme sequence. Optionally, the open reading frame is
under the control of a methanol-inducible promoter.
[0117] The present invention further provides methods for producing
an immature or mature elastase protein of the invention comprising
culturing a host cell engineered to express a nucleic acid of the
invention under conditions in which the proelastase protein is
produced. In certain embodiments, the mature elastase protein is
also produced.
[0118] Preferred culture conditions for producing the proelastase
and mature proteins of the invention, particularly for the host
cell Pichia pastoris, include a period of growth at a low pH.
Typically, the low pH is 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,
6.0, or any range between any pair of the foregoing values. In
specific embodiments, the low pH is a pH ranging from 2.0 to 6.0,
from 2.0 to 5.0, from 3.0 to 6.0, from 3.0 to 5.0, from 4.0 to 6.0,
or from 3.0 to 4.0. At the end of the culture period, the pH of the
culture can be raised, preferably to a pH of 7.0, 7.5, 8.0, 8.5,
9.0, 9.5, 10.0, 10.5, 11.0 or a pH ranging between any two of the
foregoing values for the purpose of separating or removing the
activation sequence from the mature elastase protein. In specific
embodiments, the pH of the culture is raised to a pH ranging from
7.5 to 10.0 or 8.0 to 9.0 and most preferably to a pH of 8.0.
[0119] Where the expression of a proelastase protein of the
invention is under the control of a methanol-inducible promoter,
conditions for producing an immature or mature elastase protein of
the invention may also comprise a period of methanol induction.
[0120] The elastase production methods of the invention may further
comprise the step of recovering the protein expressed by the host
cell. In certain instances, the protein recovered is a proelastase
comprising the activation sequence. In other instances, the protein
recovered is a mature elastase lacking the activation sequence.
Under certain conditions, both proelastase and mature elastase
proteins are recovered.
[0121] Preferably, particularly where it is desired to circumvent
auto-activation of an auto-activated proelastase, culture
conditions for proelastase expression may comprise a period of
growth and induction at low pH. Typically, the low pH is 2.0, 2.5,
3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, or any range between any pair
of the foregoing values. In specific embodiments, the low pH is a
pH ranging from 2.0 to 3.0, from 4.0 to 5.0, from 5.0 to 6.0, or
from 6.0 to 7.0. In particular embodiments, the pH ranges from 4.0
to 6.0 and is most preferably a pH of 5.5.
[0122] Preferably, particularly where it is desired to circumvent
auto-activation of an auto-activated proelastase, culture
conditions for proelastase expression comprise a period of growth
and induction in sodium citrate, sodium succinate, or sodium
acetate. In specific embodiments, a concentration of 5-50 mM,
7.5-100 mM, 10-15 mM, 50-200 mM, 75-175 mM, 100-150 mM, 75-125 mM,
or of any range whose upper and lower limits are selected from any
of the foregoing values (e.g., 50-75 mM, 75-100 mM, etc.) is used.
In a preferred embodiment, the sodium citrate, sodium succinate, or
sodium acetate concentration is 90-110 mM and most preferably is
100 mm.
[0123] Additionally, particularly where it is desired to circumvent
auto-activation of an auto-activated proelastase or
auto-degradation by mature elastase, culture conditions for
expression of an immature elastase protein may comprise a period of
growth and induction at the lower end of the temperature range
suitable for the host cell in question. For example, where the host
cell is a Pichia pastoris host cell, the preferred range is about
22-28.degree. C. In a specific embodiment, the Pichia pastoris host
cell is cultured at 28.degree. C.
[0124] Additionally, particularly where it is desired to circumvent
protein degradation by host cell proteases, culture conditions for
expression of an immature elastase protein may comprise a period of
growth and induction at the lower end of the temperature range
suitable for the host cell in question. For example, where the host
cell is a Pichia pastoris host cell, the preferred range is about
22-28.degree. C. In a specific embodiment, the Pichia pastoris host
cell is cultured at 28.degree. C.
[0125] The activation of an auto-activated proelastase protein of
the invention may be initiated by the addition of extrinsic
elastase in a small (catalytic) amount. In certain embodiments, a
catalytic amount of extrinsic elastase represents less than 10%,
less than 5%, less than 2%, less than 1%, less than 0.5% or less
than 0.1%, on either a molar or molecular weight basis, of the
elastase in the sample to which the catalytic elastase is
added.
[0126] Alternatively or concurrently, the auto-activated
proelastase may be subjected to pH 7-11 (most preferably pH 8),
upon which the auto-activated proelastase activation peptide is
removed without requiring trypsin and resulting in mature, active
elastase. In specific embodiments, Tris base is added to a
concentration of 50-200 mM, 75-175 mM, 100-150 mM, 75-125 mM, or
any range whose upper and lower limits are selected from any of the
foregoing values (e.g., 50-75 mM, 75-100 mM, etc.) during the
activation step. In a preferred embodiment, Tris base is added to a
concentration 90-110 mM, most preferably 100 mM. The pH of the Tris
base is preferably 7-11; in specific embodiments, the Tris base is
at a pH of 7.0-11.0, 7-9, 7.5 to 9.5, 7.5 to 10, 8-10, 8-9, or any
range whose upper and lower limits are selected from any of the
foregoing values. In a preferred embodiment, the Tris base is at a
pH of 7.5-8.5, most preferably 8.0.
[0127] Expression of an immature elastase sequence can in some
instances yield a mixture of proelastase proteins and mature
elastase proteins, as well as N-terminal variant elastase proteins.
Thus, the present invention provides a composition comprising at
least two of (1) a proelastase protein, (2) a mature elastase
protein, and (3) N-terminal variant elastase proteins.
[0128] Once a mature elastase is produced, it can be lyophilized,
for example for pharmaceutical formulations. In an exemplary
embodiment, the present invention provides methods of isolating a
lyophilized mature type I elastase comprising steps:
[0129] (a) culturing a host cell, such as a Pichia pastoris host
cell, engineered to express a nucleic acid molecule encoding a
preproelastase open reading frame under conditions in which the
open reading frame is expressed, wherein, in a specific embodiment,
said open reading frame comprises nucleotide sequences encoding, in
a 5' to 3' direction (i) a signal peptide, e.g., a signal peptide
operable in Pichia pastoris; (ii) an activation sequence comprising
an elastase recognition sequence; and (iii) the sequence of a
mature type I elastase protein, thereby producing a proelastase
protein;
[0130] (b) subjecting the proelastase protein to autoactivation
conditions, thereby producing a mature type I elastase, wherein the
autoactivation conditions include, one or a combination of the
following: [0131] (i) changing the pH of a solution (which can be a
cell culture supernatant) containing the proelastase protein, e.g.,
to a pH of 6.5-11, preferably 8-9; [0132] (ii) purifying the
proelastase protein, for example, by ion exchange chromatography,
and subjecting the solution extended conversion to remove
N-terminal variants, thereby producing mature human type I
elastase; [0133] (iii) concentrating the proelastase protein (e.g.,
2-fold, 3-fold, 5-fold, 8-fold, 10-fold, 12-fold, or a range in
which the upper and lower limits are independently selected from
the foregoing levels of concentrations); [0134] (iv) subjecting the
proelastase protein to increased temperature (e.g., 29.degree. C.,
30.degree. C., 32.degree. C., 35.degree. C., 40.degree. C.,
45.degree. C., or 40.degree. C., or a range in which the upper and
lower limits are independently selected from the foregoing
temperatures); [0135] (v) purifying the proelastase protein (e.g.,
using Macro-Prep High S Resin) from a cell culture supernatant and
incubating a solution comprising the purified proelastase protein
at ambient temperatures (e.g., 22.degree. C. to 26.degree. C.) for
a period of at least one day (e.g., one day, two days, three days,
four days five days, or six days, a range of days in which the
upper and lower limits are independently selected from the
foregoing values) (this is influenced by the presence of
citrate/acetate, concentration, temperature, and pH in the
solution, and can readily be determined by one of skill in the
art).
[0136] (c) optionally, purifying the mature human type I elastase,
e.g., ion exchange chromatography step for polish chromatography;
and
[0137] (d) lyophilizing the mature type I elastase, thereby
isolating a lyophilized mature human type I elastase. The mature
type I elastase is preferably a human type I elastase. In certain
aspects, the lyophilized mature type I elastase is preferably more
than 95% pure; in specific embodiments, the lyophilized mature type
I elastase is more than 98% or more than 99% pure.
[0138] The mature elastase proteins of the invention can be
formulated into pharmaceutical compositions. Thus, in exemplary
embodiments, the present invention provides a method of generating
a pharmaceutical composition comprising a mature human type I
elastase, said method comprising (i) isolating a lyophilized mature
human type I elastase according to the methods described above; and
(ii) reconstituting the lyophilized mature human type I elastase in
a pharmaceutically acceptable carrier. The mature human type I
elastases of the invention preferably have a specific activity of
greater than 1, greater than 5, greater than 10, greater than 20,
greater than 25, or greater than 30 U/mg of protein, as determined
by measuring the rate of hydrolysis of the small peptide substrate
N-succinyl-Ala-Ala-Ala-pNitroanilide (SLAP), which is catalyzed by
the addition of elastase. One unit of activity is defined as the
amount of elastase that catalyzes the hydrolysis of 1 micromole of
substrate per minute at 30.degree. C. and specific activity is
defined as activity per mg of elastase protein (U/mg). Preferably,
a mature human type I elastase of the invention has a specific
activity within a range in which the lower limit is 1, 2, 3, 4, 5,
7, 10, 15 or 20 U/mg protein and in which the upper limit is,
independently, 5, 10, 15, 20, 25, 30, 35, 40 or 50 U/mg protein. In
exemplary embodiments, the specific activity is in the range of
1-40 U/mg of protein, 1-5 U/mg protein, 2-10 U/mg protein, 4-15
U/mg protein, 5-30 U/mg of protein, 10-20 U/mg of protein, 20-40
U/mg of protein, or any range whose upper and lower limits are
selected from any of the foregoing values (e.g., 1-10 U/mg, 5-40
U/mg, etc.).
[0139] The pharmaceutical compositions of the invention are
preferably stable. In specific embodiments, a pharmaceutical
composition (for example a pharmaceutical composition prepared by
lyophilization and reconstitution as described above) maintains at
least 50%, more preferably at least 60%, and most preferably at
least 70% of its specific activity after a week of storage at
4.degree. C. In specific embodiments, the pharmaceutical
composition maintains at least 75%, at least 80%, at least 85% or
at least 95% of its specific activity after reconstitution and a
week of storage at 4.degree. C.
[0140] This invention also provides proteins comprising a type I
elastase proprotein amino acid sequence containing an elastase
cleavage domain sequence. Other cleavage domains that can be used
in this invention are any of the sequences described by the
concensus cleavage domain sequence (SEQ ID NO:74) Xaa.sub.1
Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6 Xaa.sub.7
Xaa.sub.8, where Xaa.sub.1=P5, Xaa.sub.2=P4, Xaa.sub.3=P3,
Xaa.sub.4=P2, Xaa.sub.5=P1, Xaa.sub.6=P'1, Xaa.sub.7=P'2, and
Xaa.sub.8=P'3, where: [0141] Xaa.sub.1 is glutamate, histidine,
proline, glycine, asparagine, lysine, or alanine, or, optionally,
an analog thereof; [0142] Xaa.sub.2 is threonine, alanine, proline
or histidine or, optionally, an analog thereof; [0143] Xaa.sub.3 is
alanine, leucine, isoleucine, methionine, lysine, asparagine or
valine, or, optionally, an analog thereof, but is preferably not
glycine or proline; [0144] Xaa.sub.4 is proline, alanine, leucine,
isoleucine, glycine, valine, or threonine, or, optionally, an
analog thereof; [0145] Xaa.sub.5 is alanine, leucine, valine,
isoleucine, or serine but not glycine, tyrosine, phenylalanine,
proline, arginine, glutamate, or lysine, or, optionally, an analog
thereof; [0146] Xaa.sub.6 is alanine, leucine, valine, isoleucine
or serine, or, optionally, an analog thereof; [0147] Xaa.sub.7 is
glycine, alanine, or valine, or, optionally, an analog thereof; and
[0148] Xaa.sub.8 is valine, threonine, phenylalanine, tyrosine, or
tryptophan, or, optionally, an analog thereof.
[0149] This invention also provides proteins comprising a type I
elastase proprotein amino acid sequence containing an elastase
cleavage domain sequence. Other cleavage domains that can be used
in this invention are any of the sequences described by the
consensus cleavage domain sequence (SEQ ID NO:123) Xaa.sub.1
Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6 Xaa.sub.7
Xaa.sub.8, where Xaa.sub.1=P5, Xaa.sub.2=P4, Xaa.sub.3=P3,
Xaa.sub.4=P2, Xaa.sub.5=P1, Xaa.sub.6=P'1, Xaa.sub.7=P'2, and
Xaa.sub.8=P'3, where: [0150] Xaa.sub.1 is glutamate, histidine,
proline, glycine, asparagine, lysine, or alanine, or, optionally,
an analog thereof; [0151] Xaa.sub.2 is threonine, alanine, proline
or histidine or, optionally, an analog thereof; [0152] Xaa.sub.3 is
alanine, leucine, isoleucine, methionine, lysine, asparagine or
valine, or, optionally, an analog thereof, but is preferably not
glycine or proline; [0153] Xaa.sub.4 is proline, alanine, leucine,
isoleucine, glycine, valine, or threonine, or, optionally, an
analog thereof; [0154] Xaa.sub.5 is alanine, leucine, valine,
isoleucine, or serine but not glycine, tyrosine, phenylalanine,
proline, arginine, glutamate, or lysine, or, optionally, an analog
thereof; [0155] Xaa.sub.6 is alanine, leucine, valine, isoleucine
or serine, or, optionally, an analog thereof; [0156] Xaa.sub.7 is
glycine, alanine, or valine, or, optionally, an analog thereof; and
[0157] Xaa.sub.8 is glycine, valine, threonine, phenylalanine,
tyrosine, or tryptophan, or, optionally, an analog thereof.
[0158] This invention also provides proteins comprising a type I
elastase proprotein amino acid sequence containing an elastase
cleavage domain sequence. Other cleavage domains that can be used
in this invention are any of the sequences described by the
consensus cleavage domain sequence (SEQ ID NO:125) Xaa.sub.1
Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6 Xaa.sub.7
Xaa.sub.8, where Xaa.sub.1=P5, Xaa.sub.2=P4, Xaa.sub.3=P3,
Xaa.sub.4=P2, Xaa.sub.5=P1, Xaa.sub.6=P'1, Xaa.sub.7=P'2, and
Xaa.sub.8=P'3, where: [0159] Xaa.sub.1 is glutamate, histidine,
proline, glycine, asparagine, lysine, or alanine, or, optionally,
an analog thereof; [0160] Xaa.sub.2 is threonine, alanine, proline
or histidine or, optionally, an analog thereof; [0161] Xaa.sub.3 is
alanine, leucine, isoleucine, methionine, lysine, asparagine,
threonine or valine, or, optionally, an analog thereof, but is
preferably not glycine or proline; [0162] Xaa.sub.4 is proline,
alanine, leucine, isoleucine, glycine, valine, asparagine, or
threonine, or, optionally, an analog thereof; [0163] Xaa.sub.5 is
alanine, leucine, valine, isoleucine, or serine but not glycine,
tyrosine, phenylalanine, proline, arginine, glutamate, asparagine,
or lysine, or, optionally, an analog thereof; [0164] Xaa.sub.6 is
alanine, leucine, valine, isoleucine or serine, or, optionally, an
analog thereof; [0165] Xaa .sub.7 is glycine, alanine, or valine,
or, optionally, an analog thereof; and [0166] Xaa.sub.8 is glycine,
valine, threonine, phenylalanine, tyrosine, or tryptophan, or,
optionally, an analog thereof.
[0167] In certain embodiments relating SEQ ID NO:74, SEQ ID NO:123,
or SEQ ID NO:125, Xaa.sub.1 is histidine and/or Xaa.sub.4 is
proline and/or Xaa.sub.5 is alanine.
[0168] This invention also provides a method of isolating a mature
human type I elastase comprising: (a) culturing, under culturing
conditions, a host cell comprising a nucleotide sequence which
encodes a proprotein comprising (i) an activation sequence
comprising a trypsin recognition sequence operably linked to (ii)
the amino acid sequence of a protein having elastase activity under
said culturing conditions, wherein said culturing conditions
comprise a period of growth or induction at pH of 2 to 6; (b)
recovering the expressed proprotein; (c) contacting the recovered
protein with a catalytic amount of trypsin under pH conditions in
which the trypsin is active; and (d) isolating mature human type I
elastase. In this method the mature human type I elastase may
consist essentially of SEQ ID NO: 1, 4, 5, 84, or 87. In certain
embodiments, the conditions may comprise (a) a period of growth or
induction at pH of 4 to 6; (b) a period of growth or induction at
22.degree. C. to 28.degree. C.; or (c) sodium citrate, sodium
succinate, or sodium acetate concentrations of about 50 mM to about
200 mM or a sodium citrate concentration is 90 mM to about 110 mM
in the culture media of said host cells.
[0169] It should be noted that the indefinite articles "a" and "an"
and the definite article "the" are used in the present application,
as is common in patent applications, to mean one or more unless the
context clearly dictates otherwise. Further, the term "or" is used
in the present application, as is common in patent applications, to
mean the disjunctive "or" or the conjunctive "and."
[0170] All publications mentioned in this specification are herein
incorporated by reference. Any discussion of documents, acts,
materials, devices, articles or the like that has been included in
this specification is solely for the purpose of providing a context
for the present invention. It is not to be taken as an admission
that any or all of these matters form part of the prior art base or
were common general knowledge in the field relevant to the present
invention as it existed anywhere before the priority date of this
application.
[0171] The features and advantages of the invention will become
further apparent from the following detailed description of
embodiments thereof.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0172] FIG. 1A-1B: FIG. 1A shows the synthetic (i.e., recombinant)
human ELA-1.2A sequence. The recombinant human elastase-1 (i.e.,
human type I pancreatic elastase) sequence contains a 750-base pair
coding region. Selected restriction enzyme sites are underlined.
Base substitutions are in double underlined, bolded text and the
codons containing them are double underlined. Stop codons are
shaded in gray (but not underlined). The propeptide sequence is
italicized. The coding region results in a 250-amino acid protein.
After cleavage of the 10-amino acid propeptide, the resulting
mature enzyme is 240 amino acids. FIG. 1B shows the pPROT24
translational fusion region. The translational fusion between the
vector and the ELA-1 coding region is depicted. The PCR
amplification of the ELA-1 sequence provided for the incorporation
of the Kex2 and STE13 signal cleavage domains to yield a secreted
product with an expected N-terminus of the first amino acid (in
bold text) of the activation sequence (italicized).
[0173] FIG. 2: A N-terminal to C-terminal schematic of the
(overlapping) core components of the elastase proteins of the
invention, wherein the numbered components depict: (1) signal
sequence (vertical stripes); (2) optional propeptide/spacer
sequence (bricks); (3) elastase propeptide (diagonal stripes, gray
fill and diamond pattern combined); (4) activation peptide (gray
fill and diamond pattern combined); (5) recognition sequence
(diamond pattern); (6) cleavage domain (gray fill, diamond pattern
and the left portion of the horizontal stripes); (7) cleavage site
(gray fill, diamond pattern and the left portion of the horizontal
stripes); (8) preproelastase protein (entire scheme); (9)
proelastase protein (diagonal stripes, gray fill, diamond pattern
and horizontal stripes combined); and (10) mature elastase protein
(horizontal stripes). The table shows the amino acid designations
of the region in the schematic spanned by the arrow. Not drawn to
scale. The nomenclature is for reference purposes only and is not
intended to connote a particular function, activity or
mechanism.
[0174] FIG. 3. Diagram of the pPROT24-V Vector. ".alpha.-factor
secretion" refers to a cassette containing the yeast .alpha.-factor
signal peptide and propeptide, followed by a Kex2 site and STE13
repeats.
[0175] FIG. 4: SDS-PAGE analysis of fractions from capture
chromatography of a 201-24-266-VU culture containing
trypsin-activated pro-PRT-201. Lane numbers correspond to fraction
numbers. Fractions 6-18 primarily consist of glycosylated proenzyme
(upper band) and non-glycosylated proenzyme (lower band). Fractions
19-35 primarily consist of non-glycosylated proenzyme. M=molecular
weight markers. FT=column flow through.
[0176] FIG. 5: Auto-activated proprotein data table. Propeptide
sequences are listed in the first column. SDS-PAGE of supernatants
after 1, 2 and 3 days of induction (lanes 1, 2 and 3, respectively)
are shown in the second column. Relative proprotein yields based on
SDS-PAGE are listed in the third column. Relative stabilities of
the proprotein over 3 days of induction based on SDS-PAGE are
listed in the fourth column. Proproteins with 42 and 48 propeptide
sequences are ranked as having low stability because of the
presence of mature protein during induction (observed after 1, 2
and 3 days for the 42 variant and after 2 and 3 days for the 48
variant). Relative conversion rates of the proproteins as
determined by time to achieve maximal SLAP reaction velocity are
listed in the fifth column. The estimated percentages of converted
protein that comprised N-terminal variants of the mature elastase
protein are listed in the sixth column.
[0177] FIG. 6. pPROT55M3-V cloning scheme. pPROT55M3-V was
engineered by in vitro ligation of two additional expression
cassettes to the 4.3 kb pPROT55-V vector backbone, giving a total
of three tandem expression cassettes. The 2.3 kb expression
cassette fragment was released from pPROT55-V with a BglII and
BamHI restriction digest and purified, followed by ligation of two
copies of the expression cassette to pPROT55-V linearized with
BamHI.
[0178] FIG. 7: Shaker flask optimization of clone 201-55M3-006-VU.
The standard induction media, BKME, was prepared and supplemented
with sodium citrate to achieve final concentrations of 0, 12.5, 25
and 50 mM sodium citrate, pH 5.5. The media was used to resuspend
growth phase cell pellets using a ratio of 1 g wet cell weight to
10 mL of induction media. Cell suspensions of 25 mL each were
placed in a 250 mL non-baffled flasks and incubated at 22.degree.
C. or 25.degree. C. for 3 days with shaking at 275 rpm. Methanol
was added twice daily to a final concentration of 0.5% by volume.
Supernatant aliquots were taken during the 3-day period and
analyzed for protein expression by SDS-PAGE and Coomassie staining.
Panel A shows samples induced at 22.degree. C. and Panel B shows
samples induced at 25.degree. C. In both panels, lanes 1-3 are
supernatants after 1, 2 and 3 days of induction, respectively,
containing 0% sodium citrate; lanes 4-6 are similar except with
12.5% sodium citrate; lanes 7-9 are similar except with 25% sodium
citrate; and lanes 10-12 are similar except with 50 mM sodium
citrate.
[0179] FIG. 8. SDS-PAGE analysis of 201-55-001-VU and
201-55M3-003-VU fermentation supernatants. Lanes 1, 2:
201-55-001-VU supernatant; lanes 3, 4: 201-55M3-003-VU supernatant;
lane 5: empty; lane 6: molecular weight markers.
[0180] FIG. 9. SDS-PAGE analysis of fractions from pPROT55M3-V
proprotein capture chromatography. A total of 30 microliters from
each elution fraction was mixed with 10 microliters 4.times.
Laemmli sample loading buffer supplemented with
beta-mercaptoethanol. The proteins were electrophoresed on an 8-16%
linear gradient gel followed by Coomassie staining. Two predominant
forms of PRT-201 were observed: the proprotein (fractions 15-43)
and spontaneously converted mature PRT-201 (fractions 15-44). Lane
numbers correspond to fraction numbers. M, molecular weight marker.
BC, before column (pre-load) sample.
[0181] FIG. 10: HIC-HPLC analysis of purified proprotein
conversion. Purified pro-PRT-201-55M3-003-VU was subjected to
conversion at 26.degree. C. The graph shows relative amounts of
mature (full-length) PRT-201 and N-terminal variants produced
during the conversion.
[0182] FIG. 11: HIC-HPLC analysis of proprotein conversion in
fermentation supernatant effected by tangential flow filtration.
Clarified 201-55M3-003-VU fermentation supernatant was subjected to
tangential flow filtration with 100 mM Tris, pH 8.0, using constant
volume diafiltration at ambient temperature with regenerated
cellulose membranes. The graph shows relative amounts of
proprotein, mature (full-length) PRT-201 and N-terminal variants
present at various time points during the conversion.
[0183] FIG. 12. SDS-PAGE analysis of fractions from pPROT55M3-V
conversion capture chromatography. A total of 30 microliters from
each elution fraction was mixed with 10 microliters 4.times.
Laemmli sample loading buffer supplemented with
beta-mercaptoethanol. The proteins were electrophoresed on an 8-16%
linear gradient gel followed by Coomassie staining. Two predominant
forms of PRT-201 were observed: the glycosylated mature form
(fractions 35-70), and the non-glycosylated mature form (fractions
75-160). Lane numbers correspond to fraction numbers. M, molecular
weight marker. BC, before column (pre-load) sample.
[0184] FIG. 13: Concentration dependence of pro conversion.
Purified pro-PRT-201 from the 201-55M3-003-VU clone
(pro-PRT-201-55M3-003-VU) was subjected to conversion at
concentrations of 0.2, 1.0, 1.6, and 1.8 mg/mL. Conversion
reactions were monitored by HIC-HPLC in real-time until the
proprotein was .ltoreq.1% of the total protein. The graph shows the
relative amounts of mature (full-length) PRT-201 (lightly shaded
bars) and N-terminal variants (darkly shaded bars) produced during
the conversions.
[0185] FIG. 14. DNA sequence of synthetic (i.e., recombinant)
porcine pancreatic elastase type 1. The recombinant sequence
contains a 750 base pair coding region. Sacll and Xbal restriction
sites as underlined were incorporated to facilitate cloning. Stop
codons are highlighted. The pro-peptide sequence is in bold-face
type.
[0186] FIG. 15. Amino acid sequence of synthetic (i.e.,
recombinant) porcine type I pancreatic elastase. The pro-peptide
region is in bold-face type while the trypsin cleavage site is
highlighted. After cleavage of the 10 amino acid pro-peptide, the
resulting mature enzyme is 240 amino acids.
[0187] FIG. 16. Cloning scheme of porcine type I pancreatic
elastase into PV-1 vector. After synthesis of the porcine type I
pancreatic elastase proprotein coding region, it was cloned in the
Blue Heron pUC vector. In addition to amplifying the coding
sequence of porcine type I pancreatic elastase, PCR was used to
incorporate XhoI and SacII restriction sites for cloning into the
PV-1 vector. The PCR product was digested, gel-purified and ligated
with PV-1 vector digested with XhoI and SacII, thus resulting in a
pPROT101-24-V expression vector encoding trypsin-activated porcine
type I pancreatic elastase proprotein.
[0188] FIG. 17. Expression analysis of auto-activated pPROT101-42-V
and trypsin-activated pPROT101-24-V clones during methanol
induction by SDS-PAGE. Shaker flask supernatants after 1 day of
induction were analyzed on an 8-16% gradient gel followed by
staining with Coomassie staining. Lanes 1-10 contain supernatants
from ten different clones transformed with pPROT101-42-V. Lanes
11-12 contain supernatants from two different clones transformed
with pPROT101-24-V. M, molecular weight markers.
[0189] FIG. 18. Expression analysis of auto-activated pPROT101-49-V
and pPROT101-55L-V clones during methanol induction by SDS-PAGE.
Shaker flask supernatants after 1 and 2 days of induction were
analyzed on an 8-16% gradient gel followed by Coomassie staining.
Lanes 1-2 contain supernatants from a pPROT101-49-V clone after 1
and 2 days of induction, respectively. Lanes 3-4 contain
supernatants from a pPROT101-55L-V clone after 1 and 2 days of
induction, respectively. M, molecular weight markers.
[0190] FIG. 19. Time course activation of pPROT101-49-V and
pPROT101-55L-V proteins by small-scale conversion assay as
determined by SLAP elastase activity. Error bars represent .+-.SD
of the mean (n=4).
[0191] FIG. 20. SDS-PAGE analysis of pPROT101-49-V and
pPROT101-55L-V supernatants before and after small-scale conversion
assay. Prior to electrophoresis, the samples were mixed with citric
acid, the reducing agent TCEP, and LDS sample buffer (Invitrogen,
Calif.). The samples were heated at 70.degree. C. for 10 minutes.
Lanes 1-2: pPROT101-49-V pre- and post-conversion assay
supernatant, respectively; lanes 3-4: pPROT101-55L-V pre- and
post-conversion assay supernatant, respectively. M, molecular
weight markers.
[0192] FIG. 21. TrypZean standard curve.
[0193] FIG. 22. A side, partially sectioned view of one embodiment
of the medical device described in Section 5.9.
[0194] FIG. 23. A view similar to FIG. 22 that illustrates the
movement of the actuators of the medical device.
[0195] FIG. 24. An end section view in the plane of line 2-2 in
FIG. 22.
[0196] FIG. 25. An end section view in the plane of line 3-3 in
FIG. 22.
[0197] FIG. 26. A diagram of the fluid path of the medical device
of FIG. 22, extending from the Luer hubs through the fluid delivery
conduits to the reservoir and then to the tissue penetrators.
[0198] FIG. 27. A side, partially sectioned view of a second
embodiment of the medical device of the present invention showing
the actuators in their constrained configurations.
[0199] FIG. 28. A view similar to FIG. 27, but showing the
actuators in their unconstrained configurations.
[0200] FIG. 29. An end perspective view of the assembly along the
line 3'-3' of FIG. 27.
[0201] FIG. 30. An end perspective view of the assembly along the
line 4'-4' of FIG. 29 showing the tissue penetrators.
[0202] FIG. 31. A side view showing the detail of the proximal end
of the device, shown to the right in FIGS. 27 and 28.
[0203] FIG. 32. A partial view of the exterior of the medical
device of FIG. 22 in its constrained position.
5. DETAILED DESCRIPTION OF THE INVENTION
[0204] The present invention is directed to methods for recombinant
expression and production of mature, biologically active elastase
proteins. The present invention provides novel, efficient methods
of making recombinant elastase proteins by culturing host cells,
including the preferred host cell, Pichia pastoris, comprising
nucleic acids encoding proelastase proteins and preproelastase
proteins. The use of the recombinant proteins to manufacture
pharmaceutical compositions for the treatment and prevention of
diseases of biological conduits (including arteries or veins) is
also provided.
[0205] In certain aspects, the present invention is directed to
recombinant auto-activated proelastase proteins and related nucleic
acids, host cells, and methods of manufacture. Such auto-activated
proelastase proteins are engineered to contain an elastase
recognition site immediately N-terminal to the first residue of the
mature elastase protein. Under specified culture conditions, such
as those described in Section 6 below, it is possible to reduce
auto-activation until activation is desired. It is also possible to
reduce auto-activation until the pro-elastase is removed from the
cell culture.
[0206] The present invention provides efficient expression and
purification processes for producing pharmaceutical grade elastase
proteins. The present invention also provides methods for treating
or preventing diseases of biological conduits using the elastase
proteins of the invention.
[0207] The description in Section 5 herein is applicable to the
embodiments of Section 8. Thus, for example, a reference to an
elastase protein of the invention includes, but is not limited to,
a reference to an elastase protein according to any one of
embodiments 1-39 and 68-69, or an elastase protein obtained or
obtainable by the method of any one of embodiments 89-224, 261 to
276, and 347 to 373. Likewise, a reference to a nucleic acid of the
invention refers, inter alia, to a nucleic acid according to any
one of embodiments 40-67; reference to a vector refers, inter alia,
a reference to a vector according to any one of embodiments 70-72;
reference to a cell refers, inter alia, to a cell according to any
one of embodiments 73-87, reference to a cell culture supernatant
refers, inter alia, to a cell culture supernatant according to
embodiment 88; reference to compositions, such as pharmaceutical
compositions, elastase formulations and unit dosages, includes, for
example, those exemplified in embodiments 277-314, 346, 386, and
415-420 or those obtained or obtainable by the method of any one of
embodiments 261-276 and 374-385; and references to therapeutic
methods also includes a reference to therapeutic methods according
to any one of embodiments 387-414; and reference to a kit includes
a reference to, inter alia, a reference to a kit of embodiments
421-425 of Section 8.
5.1 Elastase Proteins
[0208] The present invention is directed to, inter alia, methods
for recombinant expression and production of mature, biologically
active elastase proteins. The elastase proteins are generally
expressed as preproproteins, containing, among other sequences, a
signal peptide, an activation peptide, and a mature portion with
biological activity. Suitable mature elastase protein sequences are
described in Section 5.1.1 below. Suitable activation peptide
sequences are described in Section 5.1.2 below. Suitable signal
sequences are described in Section 5.1.3 below.
[0209] Accordingly, in certain aspects, the elastase proteins of
the invention are preproelastase proteins. Removal of the signal
sequence from the preproprotein upon secretion generally yields an
inactive proprotein containing an activation peptide and a mature
protein. The phrases "activation sequence" and "activation peptide"
are used interchangeably herein. Thus, in other aspects, the
elastase proteins of the invention are proelastase proteins
comprising an activation peptide that is operably linked to a
mature elastase protein. In an exemplary embodiment, an activation
peptide or sequence of a wild-type human type I pancreatic elastase
comprises the first 10 N-terminal amino acids of the human type I
elastase proprotein (SEQ ID NO:22). In certain embodiments, the
activation peptide is a peptide of SEQ ID NO:80 or SEQ ID NO:121.
Activation peptides or sequences useful in the practice of this
invention also include, but are not limited to, SEQ ID NO: 23, 72
and 73. Still other activation sequences useful in the practice of
this invention can be obtained from the N-terminal residues 1-10 of
SEQ ID NO:64-69 and 98-103.
[0210] Removal of the activation peptide from the proelastase
sequence generates a mature elastase protein. The step by which the
activation peptide is removed from the proelastase
sequence/separated from the mature elastase sequence to generate a
mature elastase protein is referred to herein as an activation
step. Thus, in yet other aspects, the elastase proteins of the
invention are mature elastase proteins.
[0211] Amino acid residues comprising the C-terminus (i.e., carboxy
terminus) of the activation peptide and the N-terminus (i.e., amino
terminus) of the mature protein that surround the cleavage bond are
depicted in FIG. 2 and also identified herein as follows. First,
residues located at the C-terminus of the activation peptide are
designated PX, . . . P5, P4, P3, P2, and P1, where P1 is the
C-terminal residue of the activation peptide. Residues located at
the N-terminus of the mature protein are designated P1', P2', P3',
. . . PX', where P1' is the N-terminal amino acid residue of the
mature protein. The scissile bond that is cleaved by proteolysis
(referred to as the "cleavage bond" in FIG. 2) is the peptide bond
between the P1 residue of the activation peptide and P1' residue of
the mature protein.
[0212] In certain preferred embodiments, a proelastase protein of
the disclosure has a histidine residue at the P5 position and/or a
proline residue at the P2 position and/or an alanine residue at the
P1 position.
[0213] The region spanning 4 amino acids of the C-terminus of the
activation peptide (residues P4 to P1) through to the first 4 amino
acids of the N-terminus of the mature protein (residues P1' to P4')
is referred to herein as the "cleavage site."
[0214] The region spanning approximately 5 amino acids of the
C-terminus of the activation peptide (i.e., residues P5 to P1)
through to approximately the first 3 amino acids of the N-terminus
of the mature protein (i.e., residues P1' to P3') is referred to
herein as the "cleavage domain." Examples of cleavage domains that
can be used in the context of this invention include, but are not
limited to, SEQ ID NOS: 42, 43, 48, 49, 52, 53, 54 or 55. Other
cleavage domains that can be used in this invention are any of the
sequences described by the consensus cleavage domain sequence
Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6
Xaa.sub.7 Xaa.sub.8, where Xaa.sub.1=P5, Xaa.sub.2=P4,
Xaa.sub.3=P3, Xaa.sub.4=P2, Xaa.sub.5=P1, Xaa.sub.6=P1',
Xaa.sub.7=P2', and Xaa.sub.8=P3', where: [0215] Xaa.sub.1 is
glutamate, histidine, proline, glycine, asparagine, lysine, or
alanine, or, optionally, an analog thereof; [0216] Xaa.sub.2 is
threonine, alanine, proline or histidine or, optionally, an analog
thereof; [0217] Xaa.sub.3 is alanine, leucine, isoleucine,
methionine, lysine, asparagine or valine, or, optionally, an analog
thereof, but is preferably not glycine or proline; [0218] Xaa.sub.4
is proline, alanine, leucine, isoleucine, glycine, valine, or
threonine, or, optionally, an analog thereof; [0219] Xaa.sub.5 is
alanine, leucine, valine, isoleucine, or serine but not glycine,
tyrosine, phenylalanine, proline, arginine, glutamate, or lysine,
or, optionally, an analog thereof; [0220] Xaa.sub.6 is alanine,
leucine, valine, isoleucine or serine, or, optionally, an analog
thereof; [0221] Xaa.sub.7 is glycine, alanine, or valine, or,
optionally, an analog thereof; and [0222] Xaa.sub.8 is glycine,
valine, threonine, phenylalanine, tyrosine, or tryptophan, or,
optionally, an analog thereof.
[0223] In specific embodiments, the proelastase cleavage domain is
any sequence described by the consensus cleavage domain sequence of
SEQ ID NO:74, SEQ ID NO:123, or SEQ ID NO:125.
[0224] In certain aspects, the proelastase proteins have a
propeptide portion of the cleavage domain of are any of the
sequences described by the consensus cleavage domain sequence
Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5, where
Xaa.sub.1=P5, Xaa.sub.2=P4, Xaa.sub.3=P3, Xaa.sub.4=P2,
Xaa.sub.5=P1, where: [0225] Xaa.sub.i is any natural amino acid;
[0226] Xaa.sub.2 is any natural amino acid except glycine, lysine,
phenylalanine, tyrosine, tryptophan, or arginine; [0227] Xaa.sub.3
is any natural amino acid; [0228] Xaa.sub.4 is proline, alanine,
leucine, isoleucine, glycine, valine, histidine, asparagine, or
threonine, and [0229] Xaa.sub.5 is alanine, leucine, valine,
isoleucine, asparagine, or serine.
[0230] In certain aspects, Xaa.sub.i is glutamate or histidine,
preferably histidine and/or Xaa.sub.4 is proline and/or Xaa.sub.5
is alanine.
[0231] The three amino acid region spanning residues P3, P2, and P1
of the activation peptide is referred to herein as an "elastase
recognition site". Examples of recognition sites that can be used
in the context of this invention include, but are not limited to,
SEQ ID NOS: 14-16, and 18-21. Other recognition sites contemplated
by this invention include any recognition site described by the
consensus recognition sites of SEQ ID NO: 11, 12, 13, or 93. The
SEQ ID NO:11 consensus elastase recognition sequence 1 is
represented by the peptide sequence Xaa.sub.1 Xaa.sub.2 Xaa.sub.3,
wherein Xaa.sub.1=P3, Xaa.sub.2=P2, Xaa.sub.3=P1, wherein: [0232]
Xaa.sub.1 is alanine, leucine, isoleucine, methionine, lysine,
asparagine or valine, or, optionally, an analog thereof but is
preferably not glycine or proline; [0233] Xaa.sub.2 is proline,
alanine, leucine, isoleucine, glycine, valine, or threonine, or,
optionally, an analog thereof; [0234] Xaa.sub.3 is alanine,
leucine, valine, isoleucine, or serine, or, optionally, an analog
thereof, but is preferably not glycine, tyrosine, phenylalanine,
proline, arginine, glutamate, or lysine.
[0235] The SEQ ID NO:12 consensus elastase recognition sequence 2
is represented by the sequence Xaa.sub.1 Pro Xaa.sub.2, wherein:
[0236] Xaa.sub.1 is alanine, leucine, isoleucine, methionine,
lysine, or valine, or, optionally, an analog thereof, but is
preferably not glycine or proline; [0237] Pro is proline, or,
optionally, an analog thereof; [0238] Xaa.sub.2 is alanine,
leucine, valine, isoleucine, or serine, or, optionally, an analog
thereof, but is preferably not glycine, tyrosine, phenylalanine,
proline, arginine, glutamate, or lysine.
[0239] The SEQ ID NO: 13 consensus elastase recognition sequence 3
is represented by the peptide sequence Xaa.sub.1 Xaa.sub.2
Xaa.sub.3, wherein Xaa.sub.1=P3, Xaa.sub.2=P2, Xaa.sub.3=P1 wherein
Xaa.sub.1 is asparagine or alanine, or, optionally, an analog
thereof, wherein Xaa.sub.2 is proline or alanine, or, optionally,
an analog thereof, and wherein Xaa.sub.3 is alanine, leucine, or
valine, or, optionally, an analog thereof.
[0240] The SEQ ID NO:93 consensus elastase recognition sequence 4
is represented by the sequence Xaa.sub.1 Pro Xaa.sub.2, wherein:
[0241] Xaa.sub.1 is alanine, leucine, isoleucine, methionine,
lysine, asparagine or valine, or, optionally, an analog thereof,
but is preferably not glycine or proline; [0242] Pro is proline,
or, optionally, an analog thereof; [0243] Xaa.sub.2 is alanine,
leucine, valine, isoleucine, or serine, or, optionally, an analog
thereof, but is preferably not glycine, tyrosine, phenylalanine,
proline, arginine, glutamate, or lysine.
[0244] The SEQ ID NO:119 consensus elastase recognition sequence 5
is represented by the sequence Xaa.sub.1 Xaa.sub.2 Xaa.sub.3,
wherein: [0245] Xaa.sub.1 is any natural amino acid, but is
preferably not glycine or proline; [0246] Xaa.sub.2 is proline,
alanine, leucine, isoleucine, glycine, valine, histidine or
threonine; [0247] Xaa.sub.3 is alanine, leucine, valine,
isoleucine, or serine.
[0248] The SEQ ID NO:124 consensus elastase recognition sequence 5
is represented by the sequence Xaa.sub.i Xaa.sub.2 Xaa.sub.3,
wherein: [0249] Xaa.sub.1 is alanine, leucine, isoleucine,
methionine, lysine, asparagine, histidine, or valine; [0250]
Xaa.sub.2 is proline, alanine, leucine, isoleucine, glycine,
valine, or threonine; [0251] Xaa.sub.3 is alanine, leucine, valine,
isoleucine, or serine.
[0252] In certain aspects, a proelastase protein of the disclosure
has a sequence for P10 through P3' described by the consensus
sequence of SEQ ID NO:122.
[0253] Reference to a sequence as a "cleavage sequence," "cleavage
domain," "activation sequence," "elastase recognition sequence,"
etc., is solely for ease of reference and is not intended to imply
any function of the sequence or mechanism by which the sequence is
recognized or processed.
[0254] The proteins of the invention are generally composed of
amino acids and may in addition include one or more (e.g., up to 2,
3, 4, 5, 6, 7, 8, 9, 10, 12 or 15) amino acid analogs. Generally,
as used herein, an amino acid refers to a naturally-occurring L
stereoisomer. An amino acid analog refers to a D-stereoisomer, a
chemically modified amino acid, or other unnatural amino acid. For
example, unnatural amino acids include, but are not limited to
azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid,
.beta.-alanine, aminopropionic acid, 2-aminobutyric acid,
4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid,
2-aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopimelic acid,
tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine,
2,2'-diaminopimelic acid, 2,3-diaminoproprionic acid,
N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine,
allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline,
isodesmosine, allo-isoleucine, N-methylalanine, N-methylglycine,
N-methylisoleucine, N-methylpentylglycine, N-methylvaline,
naphthalanine, norvaline, norleucine, ornithine, pentylglycine,
pipecolic acid and thioproline. A chemically modified amino acid
includes an amino acid that is chemically blocked, reversibly or
irreversibly, and/or modified at one or more of its side groups,
.alpha.-carbon atoms, terminal amino group, or terminal carboxylic
acid group. A chemical modification includes adding chemical
moieties, creating new bonds, and removing chemical moieties.
Examples of chemically modified amino acids include, for example,
methionine sulfoxide, methionine sulfone,
S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide
and S-(carboxymethyl)-cysteine sulfone. Modifications at amino acid
side groups include acylation of lysine .epsilon.-amino groups,
N-alkylation of arginine, histidine, or lysine, alkylation of
glutamic or aspartic carboxylic acid groups, and deamidation of
glutamine or asparagine. Modifications of the terminal amino
include the des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl
modifications. Modifications of the terminal carboxy group include
the amide, lower alkyl amide, dialkyl amide, and lower alkyl ester
modifications. A lower alkyl is a C.sub.1-C.sub.4 alkyl.
Furthermore, one or more side groups, or terminal groups, may be
protected by protective groups known to the ordinarily-skilled
protein chemist. The .alpha.-carbon of an amino acid may be mono-
or di-methylated.
[0255] The proteins of the invention may be modified or
derivatized, such as modified by phosphorylation or glycosylation,
or derivatized by conjugation, for example to a lipid or another
protein (e.g., for targeting or stabilization), or the like.
[0256] The present invention often relates to an "isolated" or
"purified" elastase protein. An isolated elastase protein is one
that is removed from its cellular milieu. A purified elastase
protein is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the elastase protein is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of elastase protein in which the protein is separated
from cellular components of the cells from which it is
recombinantly produced. Thus, elastase protein that is
substantially free of cellular material includes preparations of
elastase protein having less than about 30%, 20%, 10%, or 5% (by
dry weight) of heterologous protein (also referred to herein as a
"contaminating protein"). When the elastase protein is produced by
a process in which it is secreted into culture medium, it is also
preferably substantially free of the culture medium, i.e., culture
medium represents less than about 20%, 10%, or 5% of the volume of
the elastase protein preparation.
[0257] In certain embodiments, an isolated or purified elastase is
additionally free or substantially free of cellular DNA. In
specific embodiments, host cell genomic DNA is present in an amount
of less than 10 picogram, less than 5 picograms, less than 3
picograms, less than 2 picograms, or less than 1 picogram of DNA
per milligram of elastase protein in a preparation of isolated or
purified elastase protein, or in a composition comprising isolated
or purified elastase protein. In one embodiment, the host cell DNA
is Pichia pastoris DNA.
[0258] Useful elastase protein sequences are provided in Table 1.
In a specific embodiment, the invention provides a proelastase
protein (including but not limited to a protein of any one of SEQ
ID NOS:6-9, 64-69, 88-91 and 98-103) comprising (i) an activation
sequence comprising an elastase recognition sequence operably
linked to (ii) the amino acid sequence of a protein having type I
elastase activity. Several polymorphisms of human type I elastase
are known. Any combination of polymorphisms is contemplated in the
proelastase protein sequences of the present invention, including
but not limited to the combinations of polymorphisms set forth in
Table 2. The protein optionally further comprises a signal sequence
operably linked to said activation sequence. In certain specific
embodiments, the signal sequence is operable in Pichia pastoris,
such as a yeast .alpha.-factor signal peptide, exemplified by the
amino acid sequence of SEQ ID NO:34. Alternative signal peptide
containing sequences are exemplified in SEQ ID NOS:50 and 96
(containing a signal peptide, a non-elastase propeptide and a
spacer sequence) and SEQ ID NOS:51 and 97 (containing the signal
peptide and a non-elastase propeptide). In other specific
embodiments, the signal sequence is a mammalian secretion signal
sequence, such as a porcine elastase signal sequence. Preferably,
the elastase recognition sequence is a type I elastase recognition
sequence, most preferably a human type I elastase recognition
sequence.
[0259] The present invention further encompasses variants of the
elastase proteins of the invention. Variants may contain amino acid
substitutions at one or more predicted non-essential amino acid
residues. Preferably, a variant includes no more than 15, no more
than 12, no more than 10, no more than 9, no more than 8, no more
than 7, no more than 6, no more than 5, no more than 4, no more
than 3, no more than 2 or no more than 1 conservative amino acid
substitution relative to a naturally occurring mature elastase
and/or no more than 5, no more than 4, no more than 3, or no more
than 2 non-conservative amino acid substitutions, or no more than 1
non-conservative amino acid substitution, relative to a naturally
occurring mature elastase.
[0260] In a specific embodiments, the variant has no more than 10
or more preferably no more than five conservative amino acid
substitutions relative to a mature elastase, a proelastase or a
preproelastase of the invention, such as with respect to a mature
elastase of SEQ ID NO:1 or SEQ ID NO:84 or a proelastase protein of
SEQ ID NO:6-9, 64-69, 88-91, and 98-103. The amino acid sequences
of SEQ ID NOS:1 and 84 contain one or more positions corresponding
to potential polymorphisms in the mature elastase sequence, at
positions 44 (W or R); 59 (M or V); 220 (V or L); and 243 (Q or R)
(positions refer to preproprotein). The invention thus encompasses
mature elastase proteins with any combination of the four
polymorphisms identified in SEQ ID NO:84. Each of such combinations
is outlined in Table 3 above. The sequence of SEQ ID NOS:88-91 and
98-103 further contain a potential polymorphism in the propeptide
sequence, at position 10 (Q or H). The invention thus encompasses
preproelastase and proelastase sequences containing any combination
of the five polymorphisms identified in SEQ ID NOS:88-91 and
98-103. Each of such combinations is outlined in Table 2 above.
[0261] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan).
[0262] The variant elastase proteins of the invention may include
amino acid substitutions with amino acid analogs as well as amino
acids, as described herein.
[0263] In specific embodiments, the protein of the invention
comprises or consists essentially of a variant of a mature human
type I elastase, e.g., a variant which is at least about 75%, 85%,
90%, 93%, 95%, 96%, 97%, 98% or 99% identical to the elastase
proproteins or mature elastase proteins listed in Table 1, such as,
but not limited to, the elastase proproteins of SEQ ID NOS: 6-9,
64-69, 88-91, and 98-103, and retain elastase activity when
expressed to produce a mature elastase protein of SEQ ID NO: 1, 4,
5, 84 or 87.
[0264] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (% identity=(# of
identical positions/total # of overlapping positions).times.100).
In one embodiment, the two sequences are the same length. In other
embodiments, the two sequences differ in length by no more than 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the length of the longer
of the two sequences.
[0265] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. A preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
Such an algorithm is incorporated into the NBLAST and XBLAST
programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410.
BLAST nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to nucleic acid molecules of the invention. BLAST protein searches
can be performed with the XBLAST program, score=50, wordlength=3 to
obtain amino acid sequences homologous to protein molecules of the
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al. (1997)
Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be
used to perform an iterated search which detects distant
relationships between molecules (Id.). When utilizing BLAST, Gapped
BLAST, and PSI-Blast programs, the default parameters of the
respective programs (e.g., XBLAST and NBLAST) can be used. See
http://www.ncbi.nlm.nih.gov.
[0266] Another preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller, CABIOS (1989). Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the CGC sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Additional algorithms for sequence analysis are known
in the art and include ADVANCE and ADAM as described in Torellis
and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA
described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci.
85:2444-8. Within FASTA, ktup is a control option that sets the
sensitivity and speed of the search. If ktup=2, similar regions in
the two sequences being compared are found by looking at pairs of
aligned residues; if ktup=1, single aligned amino acids are
examined. ktup can be set to 2 or 1 for protein sequences, or from
1 to 6 for DNA sequences. The default if ktup is not specified is 2
for proteins and 6 for DNA. For a further description of FASTA
parameters, see
http://bioweb.pasteur.fr/docs/man/man/fasta.1.html#sect2, the
contents of which are incorporated herein by reference.
[0267] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, typically exact
matches are counted.
[0268] The elastase proteins of the invention can exhibit
post-translational modifications, including, but not limited to
glycosylations (e.g., N-linked or O-linked glycosylations),
myristylations, palmitylations, acetylations and phosphorylations
(e.g., serine/threonine or tyrosine). In one embodiment, the
elastase proteins of the invention exhibit reduced levels of
O-linked glycosylation and/or N-linked glycosylation relative to
endogenously expressed elastase proteins. In another embodiment,
the elastase proteins of the invention do not exhibit O-linked
glycosylation or N-linked glycosylation.
[0269] 5.1.1. The Mature Elastase Sequence
[0270] The mature elastase sequences of the present invention are
preferably mammalian elastase sequences, most preferably human
elastase sequences. In other embodiments, the mature mammalian
elastase sequences are from other mammals such as mouse, rat, pig,
cow, or horse.
[0271] In the methods and compositions of the invention, the mature
elastase sequence employed is preferably that of a type I
pancreatic elastase, which preferentially cleaves hydrophobic
protein sequences, preferable on the carboxy side of small
hydrophobic residues such as alanine. Examples of type I pancreatic
elastases include the human elastase I enzyme (NCBI Accession
Number NP.sub.--001962) that is expressed in skin and the porcine
pancreatic elastase I enzyme (NCBI Accession Number CAA27670) that
is expressed in the pancreas. SEQ ID NO:1 and SEQ ID NO:84 are
examples of mature human type I elastase sequences.
[0272] Alternatively, a type II elastase that can cleave
hydrophobic protein sequences, preferably on the carboxy side of
medium to large hydrophobic amino acid residues, may be used.
Examples of type II elastases include the human elastase IIA enzyme
(NCBI Accession Number NP254275) and the porcine elastase II enzyme
(NCBI Accession Number A26823) that are both expressed in the
pancreas.
[0273] Variants of a mature elastase protein of the invention are
also encompassed. Variants include proteins comprising amino acid
sequences sufficiently identical to or derived from the amino acid
sequence of the mature elastase protein of the invention and
exhibit elastase biological activity. A biologically active portion
of a mature elastase protein of the invention can be a protein
which is, for example, at least 150, 160, 175, 180, 185, 190, 200,
210, 220, 230, 231, 232, 233, 234, 235, 236, 237, 238, or 239 amino
acids in length. Moreover, other biologically active portions, in
which other regions of the protein are deleted, can be prepared by
recombinant techniques and evaluated for one or more of the
functional activities of the native form of a mature elastase
protein of the invention.
[0274] In addition, mature elastase proteins comprising any
combination of the four human type I elastase polymorphisms are
represented by SEQ ID NO:84. Possible combinations are set forth in
Table 3 above.
[0275] 5.1.2. Proelastase Activation Sequences
[0276] The elastase activation sequence is any sequence whose
removal from a proelastase protein results in a biologically active
mature elastase protein.
[0277] Activation sequences generally contain protease recognition
sites adjacent to where proproteins are cleaved to produce mature,
biologically active proteins. An activation sequence may be
engineered to add a protease or elastase recognition site, or it
may be engineered to replace an existing protease recognition site
with another protease recognition site. Activation peptides or
sequences useful in the practice of this invention include, but are
not limited to, SEQ ID NO: 23, 72 and 73. Still other activation
sequences useful in the practice of this invention can be obtained
from the N-terminal residues 1-10 of SEQ ID NO:64-68. In preferred
aspects, the proelastase activation sequence is engineered to
contain a recognition sequence for a type I or type II elastase.
Most preferably, the elastase recognition sequence is recognized by
the mature elastase to which it is operably linked. Thus, in
embodiments directed to a type II elastase, the recognition
sequence is most preferably a type II elastase recognition
sequence. Conversely, in embodiments directed to a type I elastase,
the recognition sequence is most preferably a type I elastase
recognition sequence. In a preferred embodiment, the recognition
sequence is a human type I elastase recognition sequence. Exemplary
type I recognition sequences include the amino acid sequence of SEQ
ID NOS: 14-16, and 18-21. Other recognition sites contemplated by
this invention include any recognition site described by the
consensus recognition sites of SEQ ID NO: 11, 12, or 13.
[0278] 5.1.3. Signal Sequences
[0279] The proelastase proteins of the invention may further
contain a signal sequence which increases the secretion of a
proelastase protein into the culture medium of the host cell in
which it is expressed.
[0280] The native signal sequence of the elastase protein may be
used, particularly for expression in a mammalian host cell. In
other embodiments, the native signal sequence of an elastase
protein of the invention can be removed and replaced with a signal
sequence from another protein, such as the porcine type I elastase
signal sequence, the human type I elastase signal sequence, or the
yeast .alpha.-factor signal sequence. In certain specific
embodiments, the yeast .alpha.-factor signal peptide can further
comprise (1) a yeast .alpha.-factor propeptide or (2) a yeast
.alpha.-factor propeptide and spacer sequence, each respectively
exemplified by the amino acid sequence of SEQ ID NOS:50 and 96 or
SEQ ID NOS:51 and 97. Alternatively, the gp67 secretory sequence of
the baculovirus envelope protein can be used as a heterologous
signal sequence (Current Protocols in Molecular Biology (Ausubel et
al., eds., John Wiley & Sons, 1992)). Other examples of
eukaryotic heterologous signal sequences include the secretory
sequences of melittin and human placental alkaline phosphatase
(Stratagene; La Jolla, Calif.). In yet another example, useful
prokaryotic heterologous signal sequences include the phoA
secretory signal (Sambrook et al., supra) and the protein A
secretory signal (Pharmacia Biotech; Piscataway, N.J.).
5.2 Elastase Nucleic Acids
[0281] One aspect of the invention pertains to recombinant nucleic
acid molecules that encode a recombinant elastase protein of the
invention. As used herein, the term "nucleic acid molecule" is
intended to include DNA molecules (e.g., cDNA or genomic DNA) and
RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated
using nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0282] The present invention is directed to nucleic acids encoding
the elastase proteins of the invention. Thus, in certain
embodiments, the present invention provides a nucleic acid molecule
comprising a nucleotide sequence which encodes a proelastase
protein (including but not limited to a protein of any one of SEQ
ID NOS:6-9, 64-69, 88-91, or 98-103) comprising (i) an activation
sequence comprising an elastase recognition sequence operably
linked to (ii) the amino acid sequence of a protein having type I
elastase activity. In other embodiments, the present invention also
provides a nucleic acid molecule comprising a nucleotide sequence
which encodes a protein comprising (i) a signal sequence operable
in Pichia pastoris operably linked to (ii) an activation sequence
(including but not limited to an amino acid sequence of SEQ ID NOS:
23, 72, or 73) comprising a protease recognition sequence which in
turn is operably linked to (iii) the amino acid sequence of a
mature human type I elastase.
[0283] A nucleic acid of the invention may be purified. A
"purified" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically
synthesized.
[0284] In instances wherein the nucleic acid molecule is a cDNA or
RNA, e.g., mRNA, molecule, such molecules can include a poly A
"tail," or, alternatively, can lack such a 3' tail.
[0285] A nucleic acid molecule of the invention can be amplified
using cDNA, mRNA or genomic DNA as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to all or a portion of
a nucleic acid molecule of the invention can be prepared by
standard synthetic techniques, e.g., using an automated DNA
synthesizer.
5.3 Recombinant Expression Vectors and Host Cells
[0286] Further provided are vectors comprising any of the nucleic
acids of the invention or host cells engineered to express the
nucleic acids of the invention. In specific embodiments, the
vectors comprise a nucleotide sequence which regulates the
expression of the protein encoded by the nucleic acid of the
invention. For example, the nucleotide sequence encoding the
protein of the invention can be operably linked to a
methanol-inducible promoter.
[0287] Host cells comprising the nucleic acids and vectors of the
invention are also provided. In certain embodiments, the vector or
nucleic acid is integrated into the host cell genome; in other
embodiments, the vector or nucleic acid is extrachromosomal. A
preferred host cell is a Pichia pastoris cell.
[0288] As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of vector is a "plasmid," which refers to
a circular double-stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector,
wherein additional DNA segments can be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors, expression vectors, are capable
of directing the expression of coding sequences to which they are
operably linked. In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids
(vectors).
[0289] The recombinant expression vectors of the invention comprise
nucleotide sequence encoding a mature elastase, a proelastase or a
preproelastase of the invention in a form suitable for expression
in a host cell. This means that the recombinant expression vectors
include one or more regulatory sequences, selected on the basis of
the host cells to be used for expression, which is operably linked
to the nucleic acid sequence to be expressed. Within a recombinant
expression vector, "operably linked" is intended to mean that the
nucleotide sequence of interest is linked to the regulatory
sequence(s) in a manner which allows for expression of the
nucleotide sequence (e.g., in an in vitro transcription/translation
system or in a host cell when the vector is introduced into the
host cell). The term "regulatory sequence" is intended to include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Such regulatory sequences are described,
for example, in Goeddel, Gene Expression Technology: Methods in
Enzymology 185 (Academic Press, San Diego, Calif., 1990).
Regulatory sequences include those which direct constitutive
expression of a nucleotide sequence in many types of host cells and
those which direct expression of the nucleotide sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). It
will be appreciated by those skilled in the art that the design of
the expression vector can depend on such factors as the choice of
the host cell to be transformed, the level of expression of
elastase protein desired, etc. The expression vectors of the
invention can be introduced into host cells to thereby produce
elastase proteins encoded by nucleic acids as described herein.
[0290] The recombinant expression vectors of the invention can be
designed for expression of an elastase protein of the invention in
prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells
(using baculovirus expression vectors), yeast cells or mammalian
cells). Suitable host cells are discussed further in Goeddel,
supra. Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0291] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of the recombinant elastase protein; 2) to
increase the solubility of the recombinant elastase protein; and 3)
to aid in the purification of the recombinant elastase protein by
acting as a ligand in affinity purification. Often, in fusion
expression vectors, a proteolytic cleavage domain is introduced at
the junction of the fusion moiety and the recombinant protein to
enable separation of the recombinant protein from the fusion moiety
subsequent to purification of the fusion protein. Thus, the fusion
moiety and proteolytic cleavage domain together can act as an
activation sequence, including a protease recognition site, for
recombinant expression of an elastase protein. Enzymes capable of
activating such fusion proteins, and their cognate recognition
sequences, include Factor Xa, thrombin and enterokinase. Typical
fusion expression vectors include pGEX (Pharmacia Biotech Inc.;
Smith and Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs,
Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant protein.
[0292] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., 1988, Gene 69:301-315) and
pET-1d (Studier et al., 1990, Gene Expression Technology: Methods
in Enzymology 185, Academic Press, San Diego, Calif., 185:60-89).
Target gene expression from the pTrc vector relies on host RNA
polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET-11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
.lamda. prophage harboring a T7 gn1 gene under the transcriptional
control of the lacUV 5 promoter.
[0293] One strategy to maximize recombinant elastase protein
expression in E. coli is to express the protein in a host bacteria
with an impaired capacity to proteolytically cleave the recombinant
protein (Gottesman, 1990, Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. 185:119-129).
Another strategy is to alter the nucleic acid sequence of the
nucleic acid to be inserted into an expression vector so that the
individual codons for each amino acid are those preferentially
utilized in E. coli (Wada et al., 1992, Nucleic Acids Res.
20:2111-2118). Such alteration of nucleic acid sequences of the
invention can be carried out by standard DNA synthesis
techniques.
[0294] In another embodiment, the expression vector is a yeast
expression vector. Examples of vectors for expression in yeast S.
cerevisiae or P. pastoris include pYepSec1 (Baldari et al, 1987,
EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, 1982, Cell
30:933-943), pJRY88 (Schultz et al., 1987, Gene 54:113-123), pYES2
(Invitrogen Corporation, San Diego, Calif.), and pPicZ (Invitrogen
Corp, San Diego, Calif.). For expression in yeast, a
methanol-inducible promoter is preferably used. Alteration of the
nucleic acid sequence of the nucleic acid to be inserted into an
expression vector so that the individual codons for each amino acid
are those preferentially utilized in P. pastoris is also
contemplated herein. More specifically, the codons of SEQ ID NO:33
or SEQ ID NO:81 can be substituted for codons that are
preferentially utilized in P. pastoris.
[0295] Alternatively, the expression vector is a baculovirus
expression vector. Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-2165) and
the pVL series (Lucklow and Summers, 1989, Virology 170:31-39).
Another strategy is to alter the nucleic acid sequence of the
nucleic acid to be inserted into an expression vector so that the
individual codons for each amino acid are those preferentially
utilized in insect cells.
[0296] In yet another embodiment, an elastase protein is expressed
in mammalian cells using a mammalian expression vector. Examples of
mammalian expression vectors include pCDM8 (Seed, 1987, Nature
329(6142):840-2) and pMT2PC (Kaufman et al., 1987, EMBO J.
6:187-195). When used in mammalian cells, the expression vector's
control functions are often provided by viral regulatory elements.
For example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40. For other
suitable expression systems for both prokaryotic and eukaryotic
cells see chapters 16 and 17 of Sambrook et al., supra.
[0297] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al., 1987, Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton,
1988, Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) and
immunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen and
Baltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle, 1989, Proc. Natl.
Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund
et al., 1985, Science 230:912-916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. EP264166).
Developmentally-regulated promoters are also encompassed, for
example the mouse hox promoters (Kessel and Gruss, 1990, Science
249:374-379) and the beta-fetoprotein promoter (Campes and
Tilghman, 1989, Genes Dev. 3:537-546).
[0298] In certain aspects of the invention, expression of a protein
of the invention may be increased by increasing the dosage of the
corresponding gene, for example by the use of a high copy
expression vector or gene amplification. Gene amplification can be
achieved in dihydrofolate reductase-("dhfr-") deficient CHO cells
by cotransfection of the gene of interest with the dhfr gene and
exposure to selective medium with stepwise increasing
concentrations of methotrexate. See, e.g., Ausubel et al., Current
Protocols in Molecular Biology Unit 16.14, (John Wiley & Sons,
New York, 1996). An alternative method for increasing gene copy
number is to multimerize an expression cassette (e.g., promoter
with coding sequence) encoding the elastase protein of interest in
a vector prior to introducing the vector into the host cell.
Methods and vectors for achieving expression cassette
multimerization are known in yeast and mammalian host cell systems
(see, e.g., Monaco, Methods in Biotechnology 8 Animal Cell
Biotechnology, at pp. 39-348 (Humana press, 1999); Vassileva et
al., 2001, Protein Expression and Purification 21:71-80; Mansur et
al., 2005, Biotechnology Letter 27(5):339-45. In addition, kits for
multi-copy gene expression are commercially available. For example,
a multi-copy Pichia expression kit can be obtained from Invitrogen
(Carlsbad, Calif.). The multimerization of an expression cassette
is exemplified in Example 6, infra.
[0299] Accordingly, other aspects of the invention pertain to host
cells into which a recombinant expression vector of the invention
has been introduced. The terms "host cell" and "recombinant host
cell" are used interchangeably herein. It is understood that such
terms refer not only to the particular subject cell but to the
progeny or potential progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term as used herein.
[0300] A host cell can be any prokaryotic (e.g., E. coli) or
eukaryotic cell (e.g., insect cells, yeast or mammalian cells).
[0301] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook et al. (supra), and other
laboratory manuals.
[0302] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a coding sequence for a selectable marker (e.g.,
for resistance to antibiotics) is generally introduced into the
host cells along with the open reading frame of interest. Preferred
selectable markers include those which confer resistance to drugs,
such as G418, hygromycin, zeocin and methotrexate. Cells stably
transfected with the introduced nucleic acid can be identified by
drug selection (e.g., cells that have incorporated the selectable
marker coding sequence will survive, while the other cells
die).
[0303] In another embodiment, the expression characteristics of an
endogenous elastase coding sequence within a cell, cell line or
microorganism may be modified by inserting a DNA regulatory element
heterologous to the elastase coding sequence into the genome of a
cell, stable cell line or cloned microorganism such that the
inserted regulatory element is operatively linked with the
endogenous gene
[0304] A heterologous sequence, containing a regulatory element,
may be inserted into a stable cell line or cloned microorganism,
such that it is operatively linked with and activates expression of
an endogenous elastase gene, using techniques, such as targeted
homologous recombination, which are well known to those of skill in
the art, and described e.g., in Chappel, U.S. Pat. No. 5,272,071;
PCT publication No. WO 91/06667, published May 16, 1991. The
heterologous sequence may further include the signal peptides,
cleavage sequences and/or activation sequences of the present
invention.
5.4 Methods of Manufacturing Mature Elastase Proteins
[0305] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce an elastase
protein of the invention. Accordingly, the invention further
provides methods for producing an elastase protein of the invention
using the host cells of the invention. In one embodiment, the
method comprises culturing the host cell of invention (into which a
recombinant expression vector encoding an elastase protein of the
invention has been introduced) in a suitable medium such that the
elastase protein is produced. In another embodiment, the method
further comprises isolating the elastase protein from the medium or
the host cell.
[0306] The present invention further provides methods for producing
immature elastase proteins of the invention comprising culturing a
host cell engineered to express a nucleic acid of the invention
under conditions in which the proelastase protein is produced. In
certain embodiments, the preproelastase protein is also produced.
The present invention further provides methods for producing mature
elastase proteins of the invention comprising culturing a host cell
engineered to express a nucleic acid of the invention under
conditions in which a proelastase protein is produced and
subjecting the proelastase protein to activation conditions such
that the mature elastase protein is produced.
[0307] Preferred culture conditions for producing the immature and
mature proteins of the invention, particularly for the host cell
Pichia pastoris, include a period of growth at a low pH. In
specific embodiments, the low pH is a pH of 2-6, a pH of 2-5, a pH
of 3-6, a pH of 3-5, a pH of 4-6, a pH of 3-4 or any range whose
upper and lower limits are selected from any of the foregoing
values. At the end of the culture period, the pH of the culture can
be raised, preferably to a pH of 7-11, most preferably to a pH of
8.
[0308] Where the expression of a proelastase protein of the
invention is under the control of a methanol-inducible promoter,
conditions for producing an immature or mature elastase protein of
the invention may also comprise a period of methanol induction.
[0309] The elastase production methods of the invention may further
comprise the step of recovering the protein expressed by the host
cell. In certain instances, the protein recovered is a proelastase,
containing the activation sequence. In other instances, the protein
recovered is a mature elastase lacking the activation sequence.
Under certain conditions, both proelastase and mature elastase
proteins are produced. In other instances, the preproelastase is
produced.
[0310] Preferably, particularly where it is desired to circumvent
auto-activation of an auto-activated proelastase, culture
conditions for proelastase expression comprise a period of growth
in sodium citrate, sodium succinate, or sodium acetate. In specific
embodiments, a concentration of about 5-50 mM, 7.5-100 Mm, 10-150
mM, 50-200 mM, 75-175 mM, 100-150 mM, 75-125 mM, or of any range
whose upper and lower limits are selected from any of the foregoing
values is used. In a preferred embodiment, the sodium citrate,
sodium succinate, or sodium acetate concentration is 90-110 mM,
most preferably 100 mM.
[0311] Additionally, particularly where it is desired to circumvent
protein degradation, culture conditions for proelastase expression
comprise a period of growth and induction at the lower end of the
temperature range suitable for the host cell in question. For
example, where the host cell is a Pichia pastoris host cell, the
preferred range is about 22-28.degree. C. In specific embodiments,
the Pichia pastoris host cell is cultured at a temperature of about
28.degree. C. The growth and induction need not be performed at the
same temperature; for example, in an embodiment where Pichia
pastoris is utilized as a host cell, growth can be performed at
28.degree. C. while induction can be performed at 22.degree. C.
[0312] The activation of an auto-activated proelastase protein of
the invention may be initiated by the addition of extrinsic
elastase in a small (catalytic) amount. Alternatively or
concurrently, the activation of an auto-activated proelastase
protein of the invention may be initiated by raising the pH of the
solution containing the auto-activated proelastase protein. The pH
is preferably 7-11; in specific embodiments, the solution is at a
pH of 7-10, 7-9,8-10, 8-9, or any range whose upper and lower
limits are selected from any of the foregoing values. In a
preferred embodiment, the pH of the solution 7-9, most preferably
8.
[0313] In specific embodiments, the auto-activated proelastase may
be subjected to Tris base, during the activation step. In specific
embodiments, Tris base is added to a concentration of 5-50 mM,
7.5-100 Mm, 10-150 mM, 50-200 mM, 75-175 mM, 100-150 mM, 75-125 mM,
or of any range whose upper and lower limits are selected from any
of the foregoing values. In a preferred embodiment, Tris base is
added to a concentration 90-110 mM, most preferably 100 mM. The pH
of the Tris base is preferably 7-11; in specific embodiments, the
Tris base is at a pH of 7-10, 7-9, 8-10, 8-9, or any range whose
upper and lower limits are selected from any of the foregoing
values. In a preferred embodiment, the Tris base is at a pH of 7-9,
most preferably 8.
[0314] In certain aspects of the invention, the temperature for
elastase autoactivation is ambient temperature, e.g., a temperature
ranging from 22.degree. C. to 26.degree. C. In certain embodiments,
the elastase activation step is preferably performed with the
proelastase at a low initial concentration, e.g., 0.1-0.3 mg/ml,
for optimal accuracy of the cleavage reaction and minimal formation
of N-terminal variants.
[0315] In certain embodiments of the invention, addition of
catalytic amounts of elastase is not required to convert the
auto-activated proelastase to mature elastase, as the proelastase
can undergo autoproteolysis. In certain embodiments, the rate of
autoproteolysis is concentration independent. Without seeking to be
limited by theory, it is believed that concentration independent
autoproteolysis of certain auto-activated proelastase proteins is
mediated via an intramolecular process where the proelastase
molecule cleaves itself via an intramolecular reaction. However, in
other embodiments, activation of the auto-activated proelastase is
concentration dependent. Without seeking to be limited by theory,
it is believed that concentration dependent autoproteolysis of
certain auto-activated proelastase proteins is mediated via an
intermolecular reaction where proelastase is cleaved by another
proelastase and/or by a mature elastase. In still other
embodiments, certain auto-activated elastase proproteins display a
combination of concentration dependent and concentration
independent activation. In those instances where auto-activation is
concentration dependent, the proprotein can be maintained in a more
dilute form to reduce activation if desired. Activation of elastase
proproteins comprising the elastase propeptide cleavage domain of
SEQ ID NO:55 that include but are not limited to the proprotein of
SEQ ID NO:69 can be controlled by maintaining such proproteins in a
dilute form. The binding cleft of mature type I elastases is
hydrophobic. Activation of elastase proproteins can also be
controlled by placing amino acids (such as histidine) in the
activation peptide that have side chains that are charged at a
lower pH but are not charged at higher pH. In this way, such
pro-proteins can be expressed and purified in low pH solutions
where activation is very slow and then activated quickly by raising
the pH to a level where elastase activity is high and the side
chain of the amino acid (such as histidine) is not charged.
[0316] It is also recognized that certain undesirable N-terminal
sequence variants of mature elastase can accumulate in the course
of producing mature elastase proteins of this invention. More
specifically, proelastase proteins containing the SEQ ID NO:42
elastase propeptide cleavage domain that include the proprotein of
SEQ ID NO:6 can yield N-terminal sequence variants where cleavage
has occurred at the peptide bond that is C-terminal to any of the
residues at positions P3 or P2. However, the occurrence of such
undesirable N-terminal variants can be reduced by placing certain
amino acids in certain locations in the activation sequence. For
example, when a proline in present in the P2 position, the
production of N-terminal variants with one or two additional
N-terminal amino acids is reduced or eliminated. Also, elimination
of the need for trypsin for activation reduces or eliminates the
production of the variant that lacks nine N-terminal residues.
Additionally, the occurrence of undesirable N-terminal variants can
be reduced or eliminated by conducting the activation reaction
under certain conditions.
[0317] More specifically, in certain embodiments activation
conditions include an "extended conversion" step during which
N-terminal variants produced during the initial portion of the
conversion reaction are subsequently selectively degraded. The
relative amounts of protein species during "extended conversion"
can be monitored in real-time by HIC-HPLC. The selective
degradation of undesired N-terminal variants increases the
proportion of full-length, mature PRT-201 in the conversion
reaction and reduces the proportion of N-terminal variants. For
proelastase proteins containing the SEQ ID NO:55 elastase
propeptide cleavage domain, the extended conversion step is
performed for 4 to 8 hours, and preferably about 6 hr. For other
proelastase proteins, the extended conversion step may be increased
or decreased, depending on the proportion of N-terminal variants
relative to mature (full length) elastase immediately after all of
the proelastase has been converted. When the conversion occurs in
complex media, such as fermentation broth, the period of extended
conversion may be increased due to competition at the active site
of mature elastase by other proteins and peptides in solution.
Alternatively, a mixture of mature elastase and N-terminal variant
elastase can be recovered from the complex media prior to the
extended conversion step, thereby reducing the active site
competition and the time required to remove the N-terminal variant
species.
[0318] As mentioned above, during a conversion reaction for
pro-PRT-201-55M3-003-VU, there is a side-reaction that leads to the
production of N-terminal variants. In the specific case of
pro-PRT-201-55M3-003-VU, these N-terminal variants are missing the
first two valines and have little or no elastase activity. For
other mutant pro-proteins there are additional N-terminal variants
produced, some with additions and others with different deletions.
An N-terminal removal step has been developed which reduces the
N-terminal variants to a range of 0-2%. The development of this
removal step evolved from a variety of experiments and
observations. As mentioned previously, during optimization of
conversion condition experiments with pro-PRT-201-42 it was
observed that longer conversion reactions often led to a very low
percentage of N-terminal variants. It was subsequently determined
that the longer conversion reactions allowed mature PRT-201 to
selectively degrade N-terminal variants. The discovery of PRT-201's
ability to selectively degrade N-terminal variants under certain
conditions had a tremendous benefit by helping to produce a more
purified PRT-201 product with less N-terminal variants. This
N-terminal variant removal step was implemented into a large scale
production process by establishing conditions that would allow the
pro-PRT-201 to convert to mature PRT-201 and then allow the PRT-201
to degrade the N-terminal variants.
[0319] A representative example of such a step is shown in FIG. 10
as it was monitored in real-time by HIC-HPLC. At approximately 50
minutes, the 100% pro-PRT-201-55M3 had completely converted to
approximately 86% mature PRT-201 and 14% N-terminal variants. The
conversion reaction was extended which allowed mature PRT-201 to
selectively degrade the N-terminal variants resulting in a decrease
of variants from 14% to 2%. With longer incubations, the N-terminal
variants can be selectively degraded to an undetectable level. When
the N-terminal variant level is sufficiently low, the activity of
PRT-201 is suppressed with sodium citrate and adjustment of the
reaction pH to 5.0.
[0320] Once purified mature elastase has been obtained, the active
enzyme can be brought into a solution at which the elastase protein
is relatively inactive and placed into a buffer for further column
chromatography, e.g., cation exchange chromatography, purification
steps. In general, the elastase protein can be placed in sodium
citrate at a concentration of about 5 to 25 mM and a pH of about 2
to 5. In a specific embodiment, the elastase is placed in 20 mM
sodium citrate, pH 5. The eluted fractions are then optionally
analyzed by one, more than one, or all of the following methods:
(1) spectrophotometry at A280 to determine concentration, (2)
SDS-PAGE to assess purity, (3) activity assay, e.g., SLAP assay, to
assess elastase specific activity, and (4) HIC-HPLC to detect
mature elastase and N-terminal variants, and fractions with
suitable characteristics (e.g., acceptable specific activity,
acceptably low levels (preferably absence) of detectable
glycoforms, and acceptably low levels (preferably absence) of
detectable N-terminal variants) are pooled.
[0321] Once purified mature elastase has been obtained, the active
enzyme can be brought into a suitable solution for lyophilization.
In general, the elastase protein can be placed into a buffer of
1.times. phosphate buffered saline ("PBS") (137 mM sodium chloride,
10 mM sodium phosphate, 2.7 mM potassium phosphate pH 7.4) prior to
lyophilization. In certain embodiments, the elastase protein can be
placed into a buffer of 0.1.times.PBS (13.7 mM sodium chloride, 1.0
mM sodium phosphate, 0.27 mM potassium phosphate pH 7.4) prior to
lyophilization.
[0322] Expression of a proelastase sequence can in some instances
yield a mixture of proelastase and mature elastase proteins. Thus,
the present invention provides a composition comprising both a
proelastase protein and a mature elastase protein.
5.5 Pharmaceutical Compositions
[0323] The mature elastase proteins of the invention can be
incorporated into pharmaceutical compositions suitable for
administration. Such compositions typically comprise the elastase
protein and pharmaceutically inert ingredients, for example a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. Also
contemplated as pharmaceutically inert ingredients are conventional
excipients, vehicles, fillers, binders, disintegrants, solvents,
solubilizing agents, and coloring agents. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with a mature elastase protein, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0324] Accordingly, certain aspects of the present invention relate
to pharmaceutical compositions. In specific embodiments, the
present invention provides a composition comprising (i) a
therapeutically effective amount of a mature human type I elastase
and (ii) a pharmaceutically acceptable carrier. The mature human
type I elastase that can be used in the composition include but are
not limited to the proteins of SEQ ID NO:1, 4, 5, 84, 87. The
mature human type I elastase may contain any of the combinations of
polymorphisms set forth in Table 3 above.
[0325] In other embodiments, the present invention provides a
pharmaceutical composition comprising (i) a therapeutically
effective amount of mature human type I elastase (ii) a
pharmaceutically acceptable carrier, which pharmaceutical
composition is free of trypsin, or fragments of trypsin. In other
embodiments, the pharmaceutical composition is substantially free
of trypsin or fragments of trypsin. As used herein, the phrase
"free of trypsin" refers to a composition in which trypsin is not
used in any portion of the production process. As used herein, the
phrase "substantially free of trypsin" refers to a composition
wherein trypsin is present at a final percent (i.e., weight
trypsin/total composition weight) of no more than about 0.0025% or
more preferably, less than about 0.001% on a weight/weight basis.
As used herein, the phrase "free of," trypsin refers to a
composition in which the variant is undetectable, e.g., by means of
an enzymatic assay or ELISA.
[0326] In certain aspects, a composition of the invention has less
trypsin activity than the equivalent of 3 ng/ml of trypsin as
measured by a BENZ assay, preferably less trypsin activity than the
equivalent of 2.5 ng/ml of trypsin as measured by a BENZ assay, and
even more preferably less trypsin activity than the equivalent of 2
ng/ml of trypsin as measured by a BENZ assay. In a specific
embodiment, the present invention provides a composition comprising
a therapeutically effective amount of elastase protein in which the
trypsin activity is the equivalent of less than 1.6 ng/ml of
trypsin as measured by a BENZ assay. Examples of elastase
compositions with less trypsin activity than the equivalent of 1.6
ng/ml of trypsin as measured by a BENZ assay are provided in
Example 8 below. In certain embodiments, the ng/ml trypsin activity
can be assayed in a liquid human type I elastase composition or
preparation containing 1 mg/ml human type I elastase protein. Thus,
the trypsin activities may also be described in terms of milligrams
of elastase protein, for example, less than 3 ng trypsin
activity/mg elastase protein, less than 1.56 ng trypsin activity/mg
elastase protein, etc.
[0327] The present invention further provides pharmaceutical
compositions that are either free or substantially free of
undesirable N-terminal variants of mature elastase. Undesirable
N-terminal variants include, but are not limited to, variants
produced by cleavage at the peptide bond that is C-terminal to any
of the residues at the P5, P4, P3, P2, P'1, P'2, P'3, P'4, P'6,
and/or P'9 positions. Certain undesirable N-terminal variants are
produced by trypsin activation; others are produced by
autoactivation of proelastase sequences that do not contained
optimized activation sequences.
[0328] In certain embodiments, the pharmaceutical composition is
free or substantially free of one, more than one or all N-terminal
variants of mature elastase that include but are not limited to SEQ
ID NOS: 2, 3, 37, 38, 70, 71, 85, 86, 94, 95, 104, 105, 106, 107,
108. In certain embodiments, the present invention provides a
pharmaceutical composition comprising (i) a therapeutically
effective amount of mature human type I elastase (ii) a
pharmaceutically acceptable carrier, which pharmaceutical
composition is free or substantially free of any protein with SEQ
ID NOS: 2, 3, 37, 38, 70, 71, 85, 86, 94, 95, 104, 105, 106, 107,
or 108. In other embodiments, the pharmaceutical composition is
substantially free of N-terminal variants of mature elastase that
include but are not limited to SEQ ID NOS: 2, 3, 37, 38, 70, 71,
85, 86, 94, 95, 104, 105, 106, 107, or 108. As used herein, the
phrase "free of" a particular variant refers to a composition in
which the variant is undetectable, e.g., by means of cation
exchange HPLC assay, hydrophobic interaction HPLC assay, or mass
spectrometry combined with liquid chromatography. As used herein,
the phrase "substantially free" refers to a composition wherein the
N-terminal variant is present at a final percent (i.e. weight
N-terminal variant/total composition weight) of at least less than
about 0.5%. In certain preferred embodiments, the composition that
is substantially free of N-terminal variant is a composition where
the concentration of N-terminal variant is less than about 0.1% or
less than about 0.01% or, more preferably, even less than about
0.001% on a weight/weight basis. In certain aspects, the presence
of N-terminal variants is detected by means of cation exchange HPLC
assay, hydrophobic interaction HPLC assay, or mass spectrometry
combined with liquid chromatography.
[0329] In certain specific embodiments, a pharmaceutical
composition that is free of N-terminal variants of SEQ ID NO:70,
71, 104 and 105 is produced by activation of a proelastase which
does not contain an arginine in the P1 position and/or an alanine
in the P2 position.
[0330] In other embodiments, the present invention provides a
pharmaceutical composition comprising (i) a therapeutically
effective amount of mature human type I elastase (ii) a
pharmaceutically acceptable carrier, which pharmaceutical
composition is substantially free of bacterial proteins and/or is
substantially free of mammalian proteins other than said mature
human type I elastase. As used herein, the phrase "substantially
free of mammalian proteins" or "substantially free of bacterial
proteins" refers to a composition wherein such proteins are present
at a final percent (i.e. weight mammalian proteins (other than
elastase and, optionally, a carrier protein such as albumin) or
bacterial proteins/total composition weight) of at least less than
about 0.5%. In certain preferred embodiments, the composition that
is substantially free of such proteins is a composition where the
concentration of the undesirable protein is less than about 0.1% or
less than about 0.01%, or, more preferably, even less than about
0.001% on a weight/weight basis.
[0331] In certain aspects, a pharmaceutical composition that is
"free of mammalian proteins" (other than elastase) contains
elastase that is produced from a recombinant cell line that is not
a mammalian cell and where no protein with a mammalian sequence or
substantially a mammalian sequence is present in any portion of the
production process. In certain aspects, a pharmaceutical
composition that is "free of bacterial proteins" contains elastase
that is produced from a recombinant cell line that is not a
bacterial cell and where no protein with a bacterial sequence or
substantially a bacterial sequence is present in any portion of the
production process.
[0332] The mature human type I elastases (including variants) of
the invention are most preferably purified for use in
pharmaceutical compositions. In specific embodiments, the elastases
are at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% pure. In
other specific embodiments, the elastases are up to 98%, 98.5%,
99%, 99.2%, 99.5% or 99.8% pure.
[0333] For formulating into pharmaceutical compositions, the mature
human type I elastases of the invention preferably have a specific
activity of greater than greater than 1, greater than 5, greater
than 10, greater than 20, greater than 25, or greater than 30 U/mg
of protein, as determined by measuring the rate of hydrolysis of
the small peptide substrate N-succinyl-Ala-Ala-Ala-pNitroanilide
(SLAP), which is catalyzed by the addition of elastase. One unit of
activity is defined as the amount of elastase that catalyzes the
hydrolysis of 1 micromole of substrate per minute at 30.degree. C.
and specific activity is defined as activity per mg of elastase
protein (U/mg). Preferably, a pharmaceutical composition comprises
a mature human type I elastase which has a specific activity within
a range in which the lower limit is 1, 2, 3, 4, 5, 7, 10, 15 or 20
U/mg protein and in which the upper limit is, independently, 5, 10,
15, 20, 25, 30, 35, 40 or 50 U/mg protein. In exemplary
embodiments, the specific activity is in the range of 1-40 U/mg of
protein, 1-5 U/mg protein, 2-10 U/mg protein, 4-15 U/mg protein,
5-30 U/mg of protein, 10-20 U/mg of protein, 20-40 U/mg of protein,
or any range whose upper and lower limits are selected from any of
the foregoing values.
[0334] The pharmaceutical compositions of the invention are
preferably stable. In specific embodiments, a pharmaceutical
composition (for example a pharmaceutical composition prepared by
lyophilization above) maintains at least 50%, more preferably at
least 60%, and most preferably at least 70% of its specific
activity after a week, more preferably after a month, yet more
preferably after 3 months, and most preferably after 6 months of
storage at 4.degree. C. In specific embodiments, the pharmaceutical
composition maintains at least 75%, at least 80%, at least 85%, at
least 90% or at least 95% of its specific activity after a week,
more preferably after a month, yet more preferably after 3 months,
and most preferably after 6 months of storage at 4.degree. C.
[0335] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration. Methods
for administering elastases to treat or prevent diseases of
biological conduits are described in WO 2001/21574; WO 2004/073504;
and WO 2006/036804. The most preferred route of administration is
parenteral, for example direct administration to the vessel wall,
including local administration to the external adventitial surface
of surgically exposed vessels and local administration to the
vessel wall using a drug delivery catheter. Solutions or
suspensions used for parenteral administration can include the
following components: a sterile diluent such as water for
injection, saline solution, phosphate buffered saline solution,
sugars such as sucrose or dextrans, fixed oils, polyethylene
glycols, glycerine, propylene glycol, polysorbate-80 (also known as
tween-80), or other synthetic solvents; antibacterial agents such
as methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampules, disposable
syringes or single or multiple dose vials made of glass or plastic.
The parenteral preparation can also be enclosed in a drug delivery
catheter. In all cases, the composition must be sterile.
[0336] In specific embodiments, a pharmaceutical composition of the
invention is a liquid formulation comprising one or more of the
following excipients: dextrose (e.g., 2-10% w/v); lactose (e.g.,
2-10% w/v); mannitol (e.g., 2-10% w/v); sucrose (e.g., 2-10% w/v);
trehalose (e.g., 2-10% w/v); ascorbic acid (e.g., 2-10 mM); calcium
chloride (e.g., 4-20 mM); dextran-70 (e.g., 2-10% w/v); poloxamer
188 (e.g., 0.2-1% w/v); polysorbate-80 (e.g., 0.001-5% w/v, more
preferably 0.1-5%); glycerin (e.g., 0.2-5% w/v); arginine (e.g.,
2-10% w/v); glycine (e.g., 2-10% w/v); dextran-44 (e.g., 2-10%
w/v); and dextran-18 (e.g., 2-10% w/v). In certain embodiments, the
concentration, singly or in the aggregate, of dextrose, lactose,
mannitol, sucrose, trehalose, dextran-70, glycerin, arginine,
glycine, dextran-44 or dextran-18 is within a range in which the
lower limit is 2.5, 4, 5, or 7% w/v and in which the upper limit
is, independently, 4, 5, 6, 8, or 10% w/v.
[0337] A liquid formulation can be made by adding water to a dry
formulation containing a mature elastase protein, one or more
buffer reagents and/or one or more excipients. The dry formulation
can be made by lyophilizing a solution comprising mature elastase
protein, one or more buffer reagents, and/or one or more
excipients.
[0338] A liquid formulation can be made, for example, by
reconstituting lyophilized elastase proteins of the invention with
sterilized water or a buffer solution. Examples of a buffer
solution include sterile solutions of saline or phosphate-buffered
saline. In a specific embodiment, after reconstitution of a dry
formulation comprising mature elastase protein to the desired
protein concentration, the solution contains approximately 137 mM
sodium chloride, 2.7 mM potassium phosphate, 10 mM sodium phosphate
(a phosphate buffered saline concentration that is considered
1.times.) and the pH of the solution is approximately 7.4. In
certain aspects, the dry formulation comprising mature elastase
protein also contains sodium, chloride, and phosphate ions in
amounts such that only water is needed for reconstitution.
[0339] A liquid formulation can be also be made, for example, by
reconstituting lyophilized elastase proteins with a buffer solution
containing one or more excipients. Examples of excipients include
polysorbate-80 and dextran. In a specific embodiment, after
reconstitution of a dry formulation comprising mature elastase
protein to the desired protein concentration, the resulting
solution contains approximately 137 mM sodium chloride, 2.7 mM
potassium phosphate, 10 mM sodium phosphate, 0.01% polysorbate-80,
and the pH of the solution is approximately 7.4. The one or more
excipients can be mixed with the mature elastase protein before
lyophilization or after lyophilization but before reconstitution.
Thus, in certain aspects, the dry formulation comprising mature
elastase protein also contains excipients such as polysorbate-80 or
dextran.
[0340] In certain aspects, the present invention provides a liquid
formulation comprising: 0.001-50 mg/ml of mature elastase protein
in a solution of 137 mM sodium chloride, 2.7 mM potassium
phosphate, 10 mM sodium phosphate and comprising 5-10%, more
preferably 6-9%, of an excipient selected from dextrose, lactose,
mannitol, sucrose, trehalose, dextran-70, glycerin, arginine,
glycine, dextran-44 or dextran-18.
[0341] In a specific embodiment, the present invention provides a
liquid formulation comprising: 0.001-50 mg/ml of mature elastase
protein in a solution of 137 mM sodium chloride, 2.7 mM potassium
phosphate, 10 mM sodium phosphate with a pH of 7.4.
[0342] In another specific embodiment, the present invention
provides a liquid formulation comprising: 0.001-50 mg/ml of mature
elastase protein in a solution of 137 mM sodium chloride, 2.7 mM
potassium phosphate, 10 mM sodium phosphate and comprising 0.01%
polysorbate-80, with a pH of 7.4.
[0343] In another specific embodiment, the present invention
provides a liquid formulation comprising: 0.001-50 mg/ml of mature
elastase protein in a solution of 137 mM sodium chloride, 2.7 mM
potassium phosphate, 10 mM sodium phosphate and comprising 0.01%
polysorbate-80 and 8% dextran-18, with a pH of 7.4.
[0344] In another specific embodiment, the present invention
provides a liquid formulation comprising: 0.001-50 mg/ml of mature
elastase protein in a solution of 137 mM sodium chloride, 2.7 mM
potassium phosphate, 10 mM sodium phosphate and comprising 8%
dextran-18, with a pH of 7.4.
[0345] The liquid formulations of the invention preferably contain
a final concentration of mature elastase proteins within a range in
which the lower limit is 0.1, 0.5, 1, 2.5, 5, 10, 15 or 20 mg/ml
and in which the upper limit is, independently, 0.5, 1, 2.5, 5, 10,
25, 50, 100, 250, 500, 1000, or 1500 mg/ml.
[0346] In certain aspects, the present invention provides a liquid
formulation comprising: 0.0001-500 mg/ml (more preferably 1-100
mg/ml, and yet more preferably 0.5-20 mg/ml) of mature elastase
protein in a solution of 0.5.times.PBS-1.5.times.PBS (more
preferably 1.times.PBS), the solution comprising 5-10% (more
preferably 6-9%) of an excipient selected from dextrose, lactose,
mannitol, sucrose, trehalose, dextran-70, glycerin, arginine,
glycine, dextran-44 or dextran-18 and having a pH of 6.5-8.5. In a
specific embodiment, the liquid formulation comprises 0.5 mg/ml
mature elastase protein and 8% dextran-18 in 1.times.PBS, pH 7.4.
In a specific embodiment, the liquid formulation comprises 5 mg/ml
mature elastase protein and 8% dextran-18 in 1.times.PBS, pH
7.4.
[0347] A liquid formulation of the invention preferably has an
osmolality within a range in which the lower limit is 100, 125,
150, 175, 200, 250 or 275 mOsm/kg and in which the upper limit is,
independently, 500, 450, 400, 350, 325, 300, 275 or 250 mOsm/kg. In
specific embodiments, the osmolarity of a liquid formulation of the
invention preferably has an osmolality of approximately 125 to 500
mOsm/kg, more preferably of approximately 275 to 325 mOsm/kg, for
example as measured by the freezing point depression method.
[0348] It is especially advantageous to formulate parenteral
compositions, such as compositions that can be made into the liquid
formulations of the invention, in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of mature elastase protein calculated to
produce the desired therapeutic effect in association with the
required pharmaceutical carrier.
[0349] As defined herein, a therapeutically effective amount of
mature elastase protein (i.e., an effective dosage) ranges from
about 0.0033 mg-200 mg. For vessels with smaller diameter and
thinner walls, such as those in a radiocephalic arteriovenous
fistula, smaller doses (such as of 0.0033 mg-2.0 mg) are
preferable. For vessels with a larger diameter and thicker walls
such as femoral arteries, larger doses (such as 2.05-100 mg) are
preferable.
[0350] In certain embodiments, the pharmaceutical compositions can
be included in a container, pack, dispenser, or catheter. In still
other embodiments, the pharmaceutical compositions can be included
in a container, pack, dispenser, or catheter together with
instructions for administration. Instructions for administration
can be included in printed form either within or upon a container,
pack, dispenser, or catheter. Alternatively, instructions for
administration can be included either within or upon a container,
pack, dispenser, or catheter in the form of a reference to another
printed or internet accessible document that provides the
instructions.
[0351] In certain embodiments, the pharmaceutical compositions can
be included in a container, pack, or dispenser. In still other
embodiments, the pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration. Instructions for administration can be included in
printed form either within or upon a container, pack, or dispenser.
Alternatively, instructions for administration can be included
either within or upon a container, pack, or dispenser in the form
of a reference to another printed or internet accessible document
that provides the instructions.
[0352] The invention includes methods for preparing pharmaceutical
compositions. Once a mature elastase is produced according to the
invention, it can be lyophilized and stored until it is
reconstituted into a pharmaceutical formulation suitable for
administration. In an exemplary embodiment, the present invention
provides a method of isolating a lyophilized mature human type I
elastase comprising: (a) culturing a host cell, such as a Pichia
pastoris host cell, engineered to express a nucleic acid molecule
encoding a preproelastase open reading frame under conditions in
which the open reading frame is expressed, wherein said open
reading frame comprises nucleotide sequences encoding, in a 5' to
3' direction (i) a signal peptide operable in Pichia pastoris; (ii)
an activation sequence comprising an elastase recognition sequence;
and (iii) the sequence of a mature type I elastase protein, thereby
producing a proelastase protein; (b) subjecting the proelastase
protein to autoactivation conditions, thereby producing a mature
type I elastase, wherein the autoactivation conditions include, for
example: (i) changing the pH of a solution containing the
proelastase protein, e.g., to a pH of 6.5-11, preferably 8-9; or
(ii) purifying the proelastase protein, for example, by ion
exchange chromatography, and subjecting the solution extended
conversion to remove N-terminal variants, thereby producing mature
human type I elastase; (c) optionally, purifying the mature human
type I elastase, e.g., ion exchange chromatography step for polish
chromatography; and (d) lyophilizing the mature type I elastase,
thereby isolating a lyophilized mature human type I elastase. The
mature type I elastase is preferably a human type I elastase. In
certain aspects, the lyophilized mature type I elastase is
preferably more than 95% pure; in specific embodiments, the
lyophilized mature type I elastase is more than 98% or more than
99% pure.
[0353] The mature elastase proteins of the invention can be
formulated into pharmaceutical compositions. Thus, in an exemplary
embodiments, the present invention provides a method of generating
a pharmaceutical composition comprising a mature human type I
elastase, said method comprising (i) isolating a lyophilized mature
human type I elastase according to the methods described above
(e.g., in Section 5.4); and (ii) reconstituting the lyophilized
mature human type I elastase in a pharmaceutically acceptable
carrier.
5.6 Effective Dose
[0354] The present invention generally provides the benefit of
parenteral, preferably local, administration of recombinant
elastase proteins, alone or in combination with other agents, for
treating or preventing disease in biological conduits.
[0355] In certain embodiments, as an alternative to parenteral
administration, oral administration of agents for treating or
preventing disease in biological conduits may be used.
[0356] Toxicity and therapeutic efficacy of the elastase proteins
utilized in the practice of the methods of the invention can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Such information can be
used to more accurately determine useful doses in humans.
5.7 Methods of Administration
[0357] The invention relates to pharmaceutical compositions
comprising novel elastase proteins and methods of use thereof for
preventing or treating disease in biological conduits. Such
pharmaceutical compositions can be formulated in a conventional
manner as described in Section 5.5 above.
[0358] The elastase compositions of the present invention can be
administered to the desired segment of the biological conduit being
treated by a device known to one of skill in the art to be
acceptable for delivery of solutions to the wall of an artery or
vein, e.g., a syringe, a drug delivery catheter, a drug delivery
needle, an implanted drug delivery polymer, such as a sheet or
microsphere preparation, an implantable catheter, or a
polymer-coated vascular stent, preferably a self-expanding
stent.
[0359] In certain embodiments, the administration to the desired
segment may be guided by direct visualization, ultrasound, CT,
fluoroscopic guidance, MRI or endoscopic guidance.
[0360] In certain aspects of the present invention, administration
of an elastase to a biological conduit comprises applying a liquid
formulation of elastase directly to the external adventitial
surface of a surgically exposed artery or vein. In specific aspects
of this invention, the administration is performed with a
syringe.
[0361] In certain aspects of the present invention, administration
of an elastase to a biological conduit comprises localizing a
delivery apparatus in close proximity to the segment of the
biological conduit to be treated. In some embodiments, during
delivery of the elastase protein by a delivery apparatus, a portion
of the delivery apparatus can be inserted into the wall of the
biological conduit. In some embodiments, the lumen of the
biological conduit can be pressurized while the elastase protein is
delivered to the pressurized segment of the biological conduit. In
some embodiments, the lumen of the biological conduit is
pressurized by mechanical action. In some embodiments, the lumen of
the biological conduit is pressurized with a balloon catheter. In
some embodiments, pressure is applied to the inner wall of the
biological conduit by a self-expanding member which is part of a
catheter or device. In some embodiments, the elastase protein is
administered and the pressurizing is performed by the same device.
In some embodiments, the biological conduit is surgically exposed
and the elastase protein is delivered into the lumen or is applied
to the external surface of the biological conduit in vivo. In
embodiments involving luminal delivery, blood flow through the
vessel may be stopped with a clamp or to allow the elastase to
contact the vessel wall for longer time periods and to prevent
inhibition of the elastase by serum. In some embodiments, the
biological conduit is surgically removed and the elastase is
delivered to the luminal surface and/or to the external surface of
the conduit in vitro. The treated conduit may then, in certain
embodiments, be returned to the body.
[0362] In other aspects of the present invention, administration of
an elastase to a biological conduit entails the use of a polymer
formulation that is placed as a stent within the vessel to be
treated, a clamp or strip applied to the external surface of the
biological conduit, or a wrap on or around the vessel to be
treated, or other device in, around or near the vessel to be
treated.
[0363] In yet other aspects of the present invention, an elastase
is percutaneously injected into a tissue region for purpose of
dilating arteries and/or vein within that region, including
collateral arteries. In other aspects, an elastase is
percutaneously injected directly into the wall of an artery or vein
or into the surrounding tissues for the purpose of dilating a
specific segment of vessel. In embodiments aimed at treatment of
heart vessels, an elastase protein can be either percutaneously
delivered to the pericardial space or directly applied to
surgically exposed coronary vessels.
[0364] Medical devices that can be used to administer the elastase
proteins of the invention to blood vessels are described in Section
5.9 below.
5.8 Kits
[0365] The present invention provides kits for practicing the
methods of the present invention. A "therapeutic" kit of the
invention comprises in one or more containers one or more of the
agents described herein as useful for treating or preventing
disease in biological conduits, optionally together with any agents
that facilitate their delivery. An alternative kit of the
invention, the "manufacturing" kit, comprises in one or more
containers one or more of the agents described herein as useful for
making recombinant elastase proteins.
[0366] The therapeutic kit of the invention may optionally comprise
additional components useful for performing the methods of the
invention. By way of example, the therapeutic kit may comprise
pharmaceutical carriers useful for formulating the agents of the
invention. The therapeutic kit may also comprise a device or a
component of a device for performing the therapeutic methods of the
invention, for example a syringe or needle. The inclusion of
devices such as an intramural or perivascular injection catheters
or intraluminal injection catheters in the therapeutic kits is also
contemplated. In certain embodiments, the agents of the invention
can be provided in unit dose form. In addition or in the
alternative, the kits of the invention may provide an instructional
material which describes performance of one or more methods of the
invention, or a notice in the form prescribed by a governmental
agency regulating the manufacture, use or sale of pharmaceuticals
or biological products, which notice reflects approval by the
agency of manufacture, use or sale for human administration.
Instructional materials can be included in printed form either
within or upon one or more containers of the kit. Alternatively,
instructional materials can be included either within or upon one
or more containers of the kit in the form of a reference to another
printed or internet accessible document that provides the
instructional materials.
[0367] In specific embodiments, a kit of the invention comprises a
medical device as described in Section 5.9 below.
[0368] The manufacturing kit of the invention may optionally
comprise additional components useful for performing the methods of
the invention.
5.9 Medical Devices Useful for Administration of Elastase
Proteins
[0369] Provided herein are medical devices that can be used to
administer the elastase proteins of the present invention to a
biological conduit, such as an artery or vein. Such devices are
described below and in provisional application No. 61/025,084,
filed Jan. 31, 2008, provisional application No. 61/025,463, filed
Feb. 1, 2008, and provisional application No. 61/075,710, filed
Jun. 25, 2008, each of which is incorporated by reference herein in
its entirety. The elastase proteins of the present invention can
also be administered to biological conduits via conventional
catheters.
[0370] In one embodiment, a medical device useful for
administration of elastase proteins has a central longitudinal
axis, and comprises one or more actuators, wherein the one or more
actuators can exist in a constrained configuration in which a
length of said one or more actuators is oriented substantially
parallel to the longitudinal axis of said medical device and an
unconstrained configuration in which at least a portion of the
length of said one or more actuators is oriented substantially
non-parallel to the device's central longitudinal axis. After the
device is positioned at a target site adjacent to the wall of a
biological conduit, one or more actuators (and if desired, all of
the actuators) may be released from a constrained configuration and
permitted to adopt an unconstrained configuration, thereby making
contact with the wall of the biological conduit. The one or more
actuators may be of any shape, and in preferred embodiments, the
movement of the one or more actuators from the constrained
configuration to the unconstrained configuration occurs upon
release of a constraining force by the device operator but without
the input by the operator of any deforming forces to the device or
the target tissue.
[0371] In a first specific embodiment, shown in FIG. 22, the device
is a fluid delivery catheter 10 comprising one or more actuators
that are formed as a pair of elongate splines 12, 14, the
intermediate regions of which are movable between a constrained
configuration which is oriented substantially parallel to the
central longitudinal axis 15 of the catheter assembly and an
unconstrained configuration in which at least a portion of the pair
of splines is oriented substantially non-parallel to said central
longitudinal axis (see the left L and right R portions of the
spline lengths in FIG. 23). The one or more splines 12, 14 may be
constructed as elongate bands or wires that each have opposite
proximal and distal ends. In a preferred embodiment, the splines
have flat, opposing interior surfaces 24, 26, and flat opposite
facing exterior surfaces 28, 30. In this embodiment, the splines
12, 14 can translate between constrained positions and
unconstrained positions, as shown respectively in FIGS. 22 and 23.
In one embodiment, the pair of splines is positioned back-to-back
in their constrained configurations as shown in FIG. 22.
[0372] The catheter 10 further comprises one or more tissue
penetrators 16, 18 secured to one or more surfaces of the one or
more splines 12, 14, a central catheter component 20 having an
elongate length, and an exterior catheter component 22 that can
shield the tissue penetrator or penetrators during catheter
movement within the biological conduit.
[0373] The tissue penetrators 16, 18 may be constructed of any
suitable material. Preferred examples of such materials include,
but are not limited to, nickel, aluminum, steel and alloys thereof.
In a specific embodiment, the tissue penetrators are constructed of
nitinol.
[0374] The central catheter component 20 and the exterior catheter
component 22 may be constructed of materials typically employed in
constructing catheters. Examples of such materials include, but are
not limited to, silicone, polyurethane, nylon, Dacron, and
PEBAX.TM.
[0375] The actuators are preferably constructed of a flexible,
resilient material. In a preferred embodiment, the flexible,
resilient material is capable of being constrained upon the
application of a constraining force, e.g., when the actuators are
in the constrained configuration, and adopts its original
unconstrained shape when the constraining force is removed, e.g.,
when the actuators are in the unconstrained configuration. Any such
flexible, resilient material can be used, including but not limited
to surgical steel, aluminum, polypropylene, olefinic materials,
polyurethane and other synthetic rubber or plastic materials. The
one or more actuators are most preferably constructed of a shape
memory material. Examples of such shape memory materials include,
but are not limited to, copper-zinc-aluminum-nickel alloys,
copper-aluminum-nickel alloys, and nickel-titanium (NiTi) alloys.
In a preferred embodiment, the shape memory material is nitinol. In
a preferred embodiment, when the pair of splines assumes the
unconstrained configuration, the shape memory properties of the
material from which each spline is formed cause the splines,
without the application of any external deforming force, to bow
radially away from each other in a single plane as shown in FIG.
23.
[0376] One or more of the splines (and preferably each of the
splines) has a flexible fluid delivery conduit 32, 34 that extends
along the length of the spline, or within the spline, as shown in
FIG. 24. As the splines 12, 14 move from their straight,
constrained configurations to their bowed, unconstrained
configurations, the fluid delivery conduits 32, 34 also move from
straight configurations to bowed configurations. In one embodiment,
the fluid delivery conduits 32, 34 are separate tubular conduits
that are secured along the lengths of the pair of splines 12, 14.
In another embodiment, the fluid delivery conduits are conduits
formed into or within the material of the splines.
[0377] One or more of the splines (and preferably each of the
splines 12,14) is also formed with a zipper rail 36, 38 that
extends along a length of the spline (FIG. 24). The zipper rails
36, 38 are formed of either the same material as the splines 12,
14, or a material that flexes with the splines 12, 14.
[0378] One or more of the tissue penetrators 16, 18 is secured to
the exterior surfaces 28, 30 of the pair of splines 12, 14 (FIG.
24). The tissue penetrators 16, 18 are connected to and communicate
with the fluid delivery conduits 32, 34 that extend along the
lengths of the splines 12, 14. The tissue penetrators 16, 18 are
positioned to project substantially perpendicular from the exterior
surfaces 28, 30 of the splines 12, 14. The tissue penetrators 16,
18 have hollow interior bores that communicate with the fluid
delivery conduits 32, 34 of the splines. The distal ends of the
tissue penetrators have fluid delivery ports that communicate with
the interior bores of the tissue penetrators.
[0379] The device permits delivery of fluids into or through one or
more distinct layers of a wall of a biological conduit, for example
a vascular wall. The vascular wall comprises numerous structures
and layers, including the endothelial layer and basement membrane
layer (collectively the intimal layer), the internal elastic
lamina, the medial layer, and the adventitial layer. These layers
are arranged such that the endothelium is exposed to the lumen of
the vessel and the basement membrane, the internal elastic lamina,
the media, and the adventitia are each successively layered over
the endothelium, as described in U.S. Pat. App. Publication No.
2006/0189941A1. With the medical devices of the present invention,
the depth to which the tissue penetrators 16, 18 can penetrate is
determined by the length of each tissue penetrator 16, 18. For
example, if the target layer is the adventitial layer, tissue
penetrators 16, 18 having a defined length sufficient for
penetration to the depth of the adventitial layer upon deployment
of the device are used. Likewise, if the target layer is the medial
layer, tissue penetrators 16, 18 having a defined length sufficient
for penetration to the depth of the medial layer upon deployment of
the device are used.
[0380] In specific embodiments, the length of tissue penetrators
16, 18 may range from about 0.3 mm to about 5 mm for vascular
applications, or up to about 20 mm or even 30 mm for applications
involving other biological spaces or conduits, for example in
colonic applications. Tissue penetrators 16, 18 preferably have a
diameter of about 0.2 mm (33 gauge) to about 3.4 mm (10 gauge),
more preferably 0.2 mm to 1.3 mm (about 33 to 21 gauge). The distal
tips of the tissue penetrators may have a standard bevel, a short
bevel, or a true short bevel. In an alternative embodiment, the
tissue penetrators attached to any one spline are not of identical
lengths, but may be configured such that their distal ends align so
as to be equidistant from the wall of the biological conduit when
the medical device is in the unconstrained position, e.g., during
use.
[0381] The central catheter component 20 has an elongate length
with opposite proximal and distal ends, shown to the left and right
respectively in FIG. 22. In one embodiment, the central catheter
component 20 has a cylindrical exterior surface that extends along
its elongate length. The proximal ends of the splines 12, 14 are
attached e.g., soldered or glued, to the distal end of the central
catheter component 20, while the distal ends of the splines 12, 14
are attached, e.g., soldered or glued, to a catheter guide tip 40.
The tip 40 has a smooth exterior surface that is designed to move
easily in the biological conduit. A guide wire bore 48 extends
through the length of the central catheter 20 and tip 40. The guide
wire bore is dimensioned to receive a guide wire in sliding
engagement through the bore.
[0382] A pair of fluid delivery lumens 44, 46 extends through the
interior of the central catheter component 20 for the entire length
of the catheter component (FIG. 25). At the distal end of the
central catheter component 20 the pair of fluid delivery lumens 44,
46 communicates with the pair of fluid delivery conduits 32, 34
that extend along the lengths of the splines 12, 14 to the tissue
penetrators 16, 18. A guide wire bore 48 also extends through the
interior of the central catheter component 20 from the proximal end
to the distal end of the central catheter component (FIG. 25). The
proximal end of the central catheter component 20 is provided with
a pair of Luer hubs 50, 52 (FIG. 22). In one embodiment, each Luer
hub 50, 52 communicates with one of the fluid delivery lumens 44,
46 extending through the length of the central catheter. Each Luer
hub 50, 52 is designed to be connected with a fluid delivery source
to communicate a fluid through each Luer hub 50, 52, then through
each fluid delivery lumen 44, 46 extending through the central
catheter component 20, then through each fluid delivery conduit 32,
34 extending along the lengths of the pair of splines 12, 14, and
then through the tissue penetrators 16, 18 secured to each of the
pair of splines. In another embodiment, each Luer hub 50, 52
independently communicates with both of the fluid delivery lumens
44, 46 extending through the length of the central catheter
component. In this configuration, a first fluid can be delivered
through a first Luer hub to both tissue penetrators 16, 18 and a
second fluid can be delivered through a second Luer hub to both
tissue penetrators 16, 18. Delivery of fluid to both tissue
penetrators from each Luer hub can be achieved by an independent
conduit extending from each Luer hub to a distal common reservoir
61 as shown in FIG. 32. This reservoir communicates with both
tissue penetrators 16, 18. Alternatively, in another embodiment,
the medical device of the instant invention comprises only a single
Luer hub connected to a single fluid delivery lumen extending
through the central catheter, which then is attached to a distal
common reservoir, permitting the delivery of a single fluid to both
tissue penetrators 16, 18.
[0383] The exterior catheter component 22 has a tubular
configuration that surrounds the pair of splines 12, 14 and a
majority of the central catheter 20 (FIG. 22). The catheter
component 22 has an elongate length that extends between opposite
proximal and distal ends of the catheter component shown to the
left and right, respectively in FIG. 22. The catheter component
distal end is dimensioned to engage in a secure engagement with the
guide tip 40, where the exterior surface of the tip 40 merges with
the exterior surface of the catheter component 22 when the catheter
component distal end is engaged with the tip. The tubular
configuration of the catheter component 22 is dimensioned so that
an interior surface of the catheter component 22 is spaced
outwardly of the plurality of tissue penetrators 16, 18 on the pair
of splines 12, 14 in the constrained positions of the pair of
splines. The proximal end of the central catheter 20 extends beyond
the proximal end of the catheter component 22 when the catheter
component distal end engages with the catheter guide tip 40.
[0384] A mechanical connection 54 is provided between the exterior
catheter component 22 proximal end and the central catheter
component 20 proximal end that enables the exterior catheter
component to be moved rearwardly along the lengths of the pair of
splines 12, 14 and the central catheter component 20 causing the
exterior catheter component 22 distal end to separate from the
guide tip 40 and pass over the pair of splines 12, 14, and
forwardly over the length of the central catheter component 20 and
over the lengths of the pair of splines 12, 14 to engage the
exterior catheter component 22 distal end with the tip 40 (FIG.
22). The mechanical connection 54 could be provided by a handle or
button that manually slides the exterior catheter component 22 over
the central catheter component 20. The connection 54 could also be
provided by a thumbwheel or trigger mechanism. In addition, the
connection 54 could be provided with an audible or tactile
indicator (such as clicking) of the incremental movement of the
exterior catheter component 22 relative to the central catheter
component 20.
[0385] In one embodiment, the exterior catheter component 22 is
provided with a single zipper track 56 that extends along the
entire length of one side of the exterior catheter component 22 on
the interior surface of the exterior catheter component (FIG. 24).
The zipper track 56 in the interior of the exterior catheter
component 22 engages in a sliding engagement with the zipper rails
36, 38 at one side of each of the splines 12, 14. Advancing the
exterior catheter component 22 forwardly along the lengths of the
central catheter component 20 and the pair of splines 12, 14 toward
the guide tip 40 of the catheter assembly causes the zipper track
56 of the exterior catheter component to slide along the rails 36,
38 of the pair of splines 12, 14. This moves the pair of splines
12, 14 from their bowed, unconstrained configuration shown in FIG.
23 toward their back-to-back, constrained configuration shown in
FIG. 22. The engagement of the spline rails 36, 38 in the zipper
track 56 of the exterior catheter component 22 holds the pair of
splines 12, 14 in their back-to-back relative positions shown in
FIG. 22. With the exterior catheter component 22 pushed forward
over the central catheter component 20 and the pair of splines 12,
14 to where the distal end of the exterior catheter component 22
engages with the guide tip 40, the tissue penetrators 16, 18 are
covered and the catheter assembly of the present invention can be
safely moved forward or backward in a biological conduit. The
exterior catheter component 22 covers the tissue penetrators 16, 18
projecting from the pair of splines 12, 14 and the engagement of
the exterior catheter component 22 with the distal guide tip 40
provides the catheter assembly with a smooth exterior surface that
facilitates the insertion of the catheter assembly into and through
a biological conduit such as a blood vessel. In another embodiment,
the exterior catheter component 22 is provided with two zipper
tracks at 180 degrees from each other that extend along the entire
length of the exterior catheter component 22 on the interior
surface and the splines have rails on both sides.
[0386] A guide wire 58 is used with the catheter assembly (FIG.
22). The guide wire 58 extends through the central catheter
component guide wire bore 48, along the splines 12, 14, and through
the guide tip outlet 42. In certain embodiments, the guide wire 58
has a solid core, e.g., stainless steel or superelastic nitinol.
The guide wire may be constructed of radiopaque material, either in
its entirety or at its distal portions (e.g., the most distal 1 mm
to 25 mm or the most distal 3 mm to 10 mm). The guide wire 58 may
optionally be coated with a medically inert coating such as
TEFLON.RTM..
[0387] In use of this device, the guide wire 58 is positioned in
the biological conduit by methods well known in the art. The guide
wire 58 extends from the biological conduit, through the guide wire
outlet 42 in the tip 40 of the assembly, through the exterior
shielding catheter 22 past the tissue penetrators 16, 18, and
through the guide wire bore 48 of the central catheter 20. In other
embodiments, the catheter assembly is a rapid-exchange catheter
assembly, wherein the guide wire lumen is present in the distal end
of the guide tip 40 of the catheter, but does not extend throughout
the entire length of the medical device.
[0388] After positioning of the guide wire, the device is advanced
into the biological conduit along the previously positioned guide
wire 58. One or more radiopaque markers may optionally be provided
on the device to monitor the position of the device in the
biological conduit. Any material that prevents passage of
electromagnetic radiation is considered radiopaque and could be
used. Preferred radiopaque materials include, but are not limited
to, platinum, gold, or silver. The radiopaque material can be
coated on the surface of all or a part of the tip 40, on all or
part of the splines 12, 14 or other actuators, on the guide wire
58, or on some combination of the foregoing structures.
Alternatively, a ring of radiopaque material can be attached to the
tip 40. The device may optionally be provided with onboard imaging,
such as intravascular ultrasound or optical coherence tomography.
The tip of the device may optionally be provided with optics that
are used to determine the position of the device or characteristics
of the surrounding biological conduit.
[0389] When the device is at its desired position in the biological
conduit, the operator uses mechanical connection 54 to retract the
exterior catheter component 22 rearwardly away from the guide tip
40. In a preferred embodiment, as the exterior catheter component
22 is withdrawn from over the tissue penetrators 16, 18, the zipper
track 56 of the exterior catheter component 22 is withdrawn over
the rails 36, 38 of the pair of splines 12, 14. This movement
releases the pair of splines 12, 14 from their constrained,
back-to-back configuration shown in FIG. 22, and allows the shape
memory material of the splines 12, 14 to adopt their unconstrained,
bowed configurations shown in FIG. 23. As the splines 12, 14 move
to their unconstrained, bowed configurations, the splines come into
contact with the inner surface of the wall(s) of the biological
conduit and the tissue penetrators 16, 18 on the exterior surfaces
28, 30 of the splines 12, 14 are pressed into the interior surface
of the biological conduit at the position of the device.
[0390] After the tissue penetrators 16, 18 have entered the desired
layer of the wall of a biological conduit, a fluid can be delivered
through the fluid delivery lumens 44, 46 in the central catheter
component 20, through the fluid delivery conduits 32, 34 on the
pair of splines 12, 14, and through the tissue penetrators 16, 18.
When the delivery of the fluid is complete, the operator uses the
mechanical connection 54 to move the exterior catheter component 22
(which may also be referred to as a shielding component) forward
over the central catheter component 20 and over the pair of splines
12, 14 toward the guide tip 40. As the exterior catheter component
22 moves forward over the pair of splines 12, 14, the zipper track
56 on the interior of the exterior catheter component 22 passes
over the rails 36, 38 on the pair of splines 12, 14, causing the
splines 12, 14 to move from their unconstrained, bowed
configuration back to their constrained configuration. When the
exterior catheter component 22 has been entirely advanced over the
pair splines 12, 14 and again engages with the guide tip 40, the
zipper track 56 in the exterior catheter component 22 holds the
splines 12, 14 in their constrained configuration. The device then
can be repositioned for release at another location in the
biological conduit or another biological conduit, or withdrawn from
the body.
[0391] The shape and length of the splines 12, 14 are selected such
that various embodiments of the device can be used in biological
spaces or conduits of various sizes or diameters. In certain
embodiments, the splines may be flat or rounded. Flat splines
preferably have a width ranging from about 0.2 mm to about 20 mm, a
height ranging from about 0.2 mm to about 5 mm, and a length
ranging from about 10 mm to about 200 mm, depending on the
particular application. Rounded splines preferably have a diameter
ranging from about 0.2 mm to about 20 mm and a length ranging from
about 10 mm to about 200 mm, depending on the particular
application. In specific embodiments, flat splines are 3.5 mm to 5
mm, 5 mm to 10 mm, 10 mm to 15 mm, 15 mm to 20 mm in width, or any
range therewithin (e.g., 3.5 mm to 10 mm); 3.5 mm to 5 mm, 5 mm to
10 mm. 10 mm to 15 mm, 15 mm to 20 mm in height, or any range
therewithin (e.g., 3.5 mm to 10 mm); and 10 mm to 20 mm, 20 mm to
40 mm, 40 mm to 80 mm, 80 mm to 120 mm, 120 mm to 150 mm or 150 to
200 mm in length, or any range therewithin (e.g., 10 mm to 40 mm),
or any permutation of the foregoing (e.g., a width of 5 mm to 10
mm, a height or 3.5 to 5 mm, and a length of 20 to 40 mm). In other
embodiments, rounded splines are 3.5 mm to 5 mm, 5 mm to 10 mm, 10
mm to 15 mm, 15 mm to 20 mm in diameter, or any range therewithin
(e.g., 3.5 mm to 10 mm) and 10 mm to 20 mm, 20 mm to 40 mm, 40 mm
to 80 mm, 80 mm to 120 mm, 120 mm to 150 mm or 150 to 200 mm in
length, or any range therewithin (e.g., 10 mm to 40 mm), or any
permutation of the foregoing (e.g., a diameter of 5 mm to 10 mm and
a length of 20 to 40 mm).
[0392] In a second specific embodiment, shown in FIG. 27, the
device of the present invention is a fluid delivery catheter 110
comprising a central catheter component 112 having an elongate
length with a longitudinal axis 113, one or more (and preferably
two) flexible, resilient actuators that, in this specific
embodiment, are formed as tissue penetrator presentation tubes 114,
116 that extend from the distal portion of the central catheter
component 112. At least a portion of the tissue presentation tubes
114, 116 are movable between a constrained configuration which is
oriented substantially parallel to the central longitudinal axis
113 of the catheter assembly and an unconstrained configuration
which is oriented substantially non-parallel to the central
longitudinal axis 113 of the catheter.
[0393] The catheter further comprises one or more (and preferably
two) flexible, elongate tissue penetrators 118, 120 that extend
through the two tissue penetrator presentation tubes 114, 116, and
an exterior deployment tube 122 that extends over portions of the
lengths of the central catheter component 112, the tissue
penetrator presentation tubes 114, 116, and the middle rail
132.
[0394] The central catheter component 112 and the exterior
deployment tube 122 may be constructed of any materials suitable
for constructing catheters. Examples of such materials include, but
are not limited to, silicone, polyurethane, nylon, Dacron, and
PEBAX.TM..
[0395] The tissue penetrators 118, 120 connect to respective hubs
166, 168 (FIG. 31). One or more of the pair of tissue penetrators
118, 120 preferably has a diameter of about 0.2 mm (33 gauge) to
about 3.4 mm (10 gauge), more preferably 0.8 mm to 1.3 mm (about 18
to 21 gauge). One or more of the pair of tissue penetrators may
have a standard bevel, a short bevel or a true short bevel. The
pair of tissue penetrators 118, 120 are preferably constructed of
materials that allow the tissue penetrators to flex along their
lengths. Examples of such materials include, but are not limited
to, nickel, aluminum, steel and alloys thereof. In a specific
embodiment, the tissue penetrators are constructed of nitinol. The
full length of the tissue penetrators 118, 120 can be constructed
of a single material, or the distal ends (e.g., the distal 1 mm to
the distal 20 mm), including the tips 156, 158, of the tissue
penetrators 118, 120 may be constructed of one material and
connected to the respective hubs 166, 168 via a tubing constructed
of a different material, e.g., plastic.
[0396] One or more of the pair of tissue penetrator presentation
tubes 114, 116 is preferably constructed of a flexible, resilient
material. Such flexible, resilient material can be deformed, e.g.,
when the tissue penetrator presentation tubes 114, 116 are in the
straight, constrained configuration of FIG. 27, but returns to its
original shape when the deformation force is removed, e.g., when
the tissue penetrator presentation tubes 114, 116 are in the
curved, unconstrained configuration shown in FIG. 28. Any such
flexible, resilient material can be used, including but not limited
to surgical steel, aluminum, polypropylene, olefinic materials,
polyurethane and other synthetic rubber or plastic materials. The
pair of tissue penetrator presentation tubes 114, 116 is most
preferably constructed of a shape memory material. Examples of such
shape memory materials include, but are not limited to,
copper-zinc-aluminum-nickel alloys, copper-aluminum-nickel alloys,
and nickel-titanium (NiTi) alloys. In a preferred embodiment, the
shape memory material is nitinol.
[0397] The central catheter component 112 has a flexible elongate
length with opposite proximal 124 and distal 126 ends (FIG. 27).
The distal end 126 of the central catheter component is formed as a
guide tip that has an exterior shape configuration that will guide
the distal end 126 through a biological conduit. A guide wire bore
128 within middle rail 132 extends through the center of the
central catheter 112 from the proximal end 124 to the distal end
126. The guide wire bore 128 receives a flexible, elongate guide
wire 130 for sliding movement of the bore 128 over the wire (FIG.
29). The guide wire 130 is used to guide the catheter assembly
through a biological conduit. In certain embodiments, the guide
wire 130 has a solid core, e.g., stainless steel or superelastic
nitinol. The guide wire may optionally be constructed of radiopaque
material, either in its entirety or at its distal portions (e.g.,
the most distal 1 mm to 25 mm or the most distal 1 mm to 3 mm). The
guide wire 130 may optionally be coated with a medically inert
coating such as TEFLON.RTM.. In other embodiments, the catheter
assembly is a rapid-exchange catheter assembly wherein a guide wire
is positioned on the distal end of the guide tip 126 and extends
therefrom.
[0398] A narrow middle rail 132 surrounding the guide wire bore 128
extends from the guide tip of the catheter distal end 126 toward
the catheter proximal end 124. The middle rail 132 connects the
guide tip 126 to a base portion 138 of the central catheter
component.
[0399] The central catheter component base portion 138 has a
cylindrical exterior surface that extends along the entire length
of the base portion. The base portion 138 extends along a majority
of the overall length of the central catheter component 112. As
shown in FIG. 29, the guide wire bore 128 extends through the
center of the central catheter component base portion 138. In
addition, a pair of tissue penetrator lumens 140, 142 also extend
through the length of the central catheter component base portion
138 alongside the guide wire bore 128. At the proximal end 124 of
the central catheter component, a pair of ports 144, 146
communicate the pair of lumens 140, 142 with the exterior of the
central catheter component 112 (FIG. 27).
[0400] In an alternative embodiment, the medical device of FIG. 27
also may comprise a single flexible, resilient actuator that is
formed as a tissue penetrator presentation tube, a single flexible,
elongate tissue penetrator that extends through the tissue
penetrator presentation tube and connects to a hub, and an exterior
deployment tube that extends over portions of the lengths of the
central catheter component, the tissue penetrator presentation
tube, and the middle rail.
[0401] The pair of first and second tissue penetrator presentation
tubes 114, 116 project from the catheter central component base
portion 138 toward the catheter distal end 126. Each of the tissue
penetrator presentation tubes is formed as a narrow, elongate tube
having a proximal end that is secured to the central catheter
component base portion 138, and an opposite distal end 148, 150.
Each of the first and second tissue penetrator presentation tubes
114, 116 has an interior bore 152, 154 that communicates with the
respective first tissue penetrator lumen 140 and second tissue
penetrator lumen 142 in the central catheter component base portion
138.
[0402] As shown in FIGS. 29 and 30, the exterior configurations of
the tissue penetrator presentation tubes 114, 116 are matched to
the middle rail 132 so that the lengths of the tissue penetrator
presentation tubes 114, 116 may be positioned side-by-side on
opposite sides of the middle rail 132. The tissue penetrator tube
distal ends 148, 150 can be formed as guide tip surfaces that also
facilitate the passage of the catheter through a vascular system.
The tissue penetrator tube distal ends 148, 150 are preferably
larger in diameter than the tissue penetrator presentation tubes
114, 116. In a specific embodiment, the tissue penetrator tube
distal tips 148, 150 are rounded and bulbous tips. Such tips are
atraumatic and the tubes will not inadvertently puncture the wall
of a biological conduit. The tips 148, 150 are exposed and do not
extend outwardly beyond the diameter of the guide tip 126.
[0403] Each of the tissue penetrator tubes 114, 116 is preferably
constructed of a shape memory material, such as nitinol. The tubes
114, 116 are formed with curved, unconstrained configurations shown
in FIG. 28. The tubes 114, 116 move to the curved, unconstrained
configurations shown in FIG. 28 when no constraining force is
applied against the tubes. In order for the presentation tubes 114,
116 to lie in straight, constrained configurations along the middle
rail 132, a constraining force must be applied to the tubes to keep
them in their straight, constrained positions shown in FIG. 27. As
each of the tubes 114, 116 moves from its straight, constrained
configuration shown in FIG. 28 to its curved, unconstrained
configuration shown in FIG. 28, the tissue penetrator bores 152,
154 extending through the tubes also move from straight
configurations to curved configurations.
[0404] The pair of tissue penetrators 118, 120, from their distal
tips to the hubs 166, 168, have lengths that are slightly longer
than the combined lengths of the tissue penetrator lumens 140, 142
extending through the central catheter base portion 138 and the
tissue penetrator bores 152, 154 extending through the tissue
penetrator presentation tubes 114, 116. The tips 156, 158 of the
tissue penetrators 118, 120 are positioned adjacent to the distal
ends 148, 150 of the tissue penetrator presentation tubes 114, 116
and are positioned inside of the bores 152, 154 of the tubes in the
constrained configuration of FIG. 27. The opposite, proximal ends
of the tissue penetrators 118, 120 project out through the side
ports 144, 146 of the central catheter 112. The pair of tissue
penetrators 118, 120 are dimensioned to easily slide through the
tissue penetrator lumens 140, 142 of the central catheter component
112 and the tissue penetrator bores 152, 154 of the tissue
penetrator presentation tubes 114, 116. The side ports 144, 146 of
the central catheter component 112 are preferably at 20.degree. to
90.degree. angles to the central catheter proximal end 124, most
preferably at 30.degree. to 60.degree. angles to the central
catheter proximal end 124.
[0405] A pair of manual operator movement to linear movement
controllers 162, 164 can be connected to the proximal ends of the
tissue penetrators 118, 120 and can be secured to the central
catheter ports 144, 146 (FIG. 31). The controllers 162, 164 can be
constructed to convert operator movement into controlled linear
movement of the tissue penetrators 118, 120 through the central
catheter tissue penetrator lumens 140, 142 and through the tissue
penetrator presentation tube bores 152, 154. In one embodiment,
there are rotating controllers 162, 164 that can be manually moved
in one direction, such that the tissue penetrator injection tips
156, 158 at the tissue penetrator distal ends can be adjustably
positioned to extend a desired length out from the tissue
penetrator tube bores 152, 154 at the tissue penetrator tube distal
ends 148, 150. By rotating the controllers in the opposite
direction, the tissue penetrators 118, 120 can be retracted back
into the tissue penetrator tube bores 152, 154. Each of the
operator movement to linear movement controllers 162, 164 can be
provided with a hub 166, 168 that communicates with the interior
bore extending through the tissue penetrators 118, 120 and can be
used to connect a syringe or tubing containing a solution of a
diagnostic or therapeutic agent.
[0406] The exterior deployment tube 122 has a tubular length that
surrounds the central catheter 112, the tissue penetrator
presentation tubes 114, 116, and the middle rail 132. The
deployment tube 122 can be mounted on the central catheter
component 112 and the pair of tissue penetrator presentation tubes
114, 116 for sliding movement to a forward position of the
deployment tube 122 where an open distal end 172 of the deployment
tube is positioned adjacent the distal ends 148, 150 of the tissue
penetrator presentation tubes 114, 116 as shown in FIG. 27, and a
rearward position of the deployment tube 122 where the tube distal
end 172 is positioned adjacent to the connection of the tissue
penetrator presentation tubes 114, 116 with the central catheter
component 112 as shown in FIG. 28. The opposite proximal end 174 of
the deployment tube 122 can be provided with a mechanical
connection 176 to the central catheter 112. The mechanical
connection 176 enables the deployment tube 122 to be moved between
its forward and rearward positions relative to the central catheter
112 and the tissue penetrator presentation tubes 114, 116 (FIGS. 27
and 28). Such a connection could be provided by a thumbwheel, a
sliding connection, a trigger or push button or some other
connection that is manually operable to cause the deployment tube
122 to move relative to the central catheter 112 and the
presentation tubes 114, 116. When the deployment tube 122 is moved
to its forward position shown in FIG. 27, the tube distal end 172
passes over the lengths of the tissue penetrator presentation tubes
114, 116 and moves the presentation tubes to their constrained
positions extending along the opposite sides of the central
catheter middle rail 132. When the deployment tube 122 is moved to
its rearward position shown in FIG. 28, the distal end 172 of the
deployment tube is retracted from over the length of the tissue
penetrator presentation tubes 114, 116 and gradually allows the
presentation tubes 114, 116 to release their constrained energy and
move to their curved, unconstrained configurations shown in FIG.
28.
[0407] In use of the catheter 110, the deployment tube 122 is in
the forward position shown in FIG. 27. The guide wire 130 is
positioned in a biological conduit (such as an artery or vein) in a
known manner. The catheter is then advanced into the biological
conduit over the guide wire. The guide wire 130 extends from the
biological conduit, and enters the central catheter component
distal end 126 through the guide wire lumen 128. The wire 130
passes through the length of the central catheter 112 and emerges
at the proximal end of the central catheter component adjacent to
the catheter ports 144, 146, where the guide wire 130 can be
manually manipulated.
[0408] The catheter 110 can be advanced through the biological
conduit and can be guided by the guide wire 130. Radiopaque markers
may optionally be provided on the assembly to monitor the position
of the assembly in the biological conduit. Any material that
prevents passage of electromagnetic radiation is considered
radiopaque and may be used. Useful radiopaque materials include,
but are not limited to, platinum, gold, or silver. The radiopaque
material can be coated on the surface of all or a part of the tip
126, on all or part of the presentation tubes 114, 116, on all or
part of the tissue penetrators 118, 120, on the guide wire 130, or
on any combination of the foregoing structures. Alternatively, a
ring of radiopaque material can be attached to the tip 126. The
assembly may optionally be provided with onboard imaging, such as
intravascular ultrasound or optical coherence tomography. The tip
of the assembly may optionally be provided with optics that are
useful for determining the position of the assembly or the
characteristics of the surrounding biological conduit. When the
assembly is at a desired position, the exterior deployment tube 122
can be moved from its forward position shown in FIG. 27 toward its
rearward position shown in FIG. 28 by manual manipulation of the
mechanical connection 176.
[0409] As the deployment tube 122 is withdrawn from over the pair
of tissue penetrator presentation tubes 114, 116, the constrained
energy of the tissue penetrator presentation tubes 114, 116 is
released and the tubes move toward their unconstrained, curved
configurations shown in FIG. 28. This movement positions the tissue
penetrator bores 152, 154 at the tissue penetrator tube distal ends
148, 150 against the interior surfaces of the biological conduit
into which the assembly 110 has been inserted.
[0410] The operator movement to linear movement controllers 162,
164 then can be manually operated to extend the tissue penetrator
distal ends 156, 158 from the tissue penetrator bores 152, 154 at
the tissue penetrator presentation tube distal ends 148, 150. A
gauge may be provided on each of the operator movement to linear
movement controllers 162, 164 that provides a visual indication of
the extent of the projection of the tissue penetrator tips 156, 158
from the tissue penetrator tube ends 148, 150 as the controllers
162, 164 are rotated. The controllers also could provide an audible
sound or tactile feel such as clicking to indicate incremental
distance steps of the tissue penetrator movements. This deploys the
tissue penetrator tips 156, 158 a desired distance into the walls
of the biological conduit.
[0411] In a third specific embodiment, a medical device of the
instant invention is a fluid delivery catheter comprising one or
more tissue penetrators constructed of a flexible, resilient
material. In certain aspects, the medical device of the present
invention has a central longitudinal axis, and comprises one or
more tissue penetrators, wherein the one or more tissue penetrators
can exist in a constrained configuration in which a length of said
one or more tissue penetrators is oriented substantially parallel
to the longitudinal axis of said medical device and an
unconstrained configuration in which at least a portion of the
length of said one or more tissue penetrators is oriented
substantially non-parallel to the device's central longitudinal
axis. After the device is positioned at a target site adjacent to
the wall of a biological conduit, one or more tissue penetrators
(and if desired, all of the tissue penetrators) may be released
from a constrained configuration and permitted to adopt an
unconstrained configuration, thereby making contact with the wall
of the biological conduit. The one or more tissue penetrators may
be of any shape, and in preferred embodiments, the movement of the
one or more tissue penetrators from the constrained configuration
to the unconstrained configuration occurs upon release of a
constraining force by the device operator but without the input by
the operator of any deforming forces to the device or the target
tissue.
[0412] In a preferred embodiment, tissue penetrators are
constructed of flexible, resilient material that is capable of
being constrained upon the application of a constraining force,
e.g., when the tissue penetrators are in the constrained
configuration, and adopts its original unconstrained shape when the
constraining force is removed, e.g., when the tissue penetrators
are in the unconstrained configuration. Any such flexible,
resilient material can be used, including but not limited to
surgical steel, aluminum, polypropylene, olefinic materials,
polyurethane and other synthetic rubber or plastic materials. The
one or more tissue penetrators are most preferably constructed of a
shape memory material. Examples of such shape memory materials
include, but are not limited to, copper-zinc-aluminum-nickel
alloys, copper-aluminum-nickel alloys, and nickel-titanium (NiTi)
alloys. In a preferred embodiment, the shape memory material is
nitinol. In a preferred embodiment, when the tissue penetrators
assume the unconstrained configuration, the shape memory properties
of the material from which each tissue penetrator is formed cause
the tissue penetrators, without the application of any external
deforming force, to move from a position substantially parallel to
the longitudinal axis of the medical device to a position
substantially perpendicular to the longitudinal axis of the medical
device.
[0413] In a preferred embodiment, the tissue penetrators are
maintained in the constrained configuration by an exterior catheter
component having a tubular configuration that surrounds the tissue
penetrators. A mechanical connection is provided between the
exterior catheter component and the central catheter component to
which the tissue penetrators are attached. The mechanical
connection enables the exterior catheter component to be moved
rearwardly along the length of the central catheter component,
thereby uncovering the constrained one or more tissue penetrators
and permitting the one or more tissue penetrators to assume an
unconstrained configuration wherein they make contact with the
target delivery site. One of ordinary skill in the art would
appreciate that this specific embodiment may be readily adapted to
incorporate radiopaque markers to facilitate positioning of the
device or rapid-exchange features to facilitate the use of the
device.
[0414] The medical device of the present invention, in its various
embodiments, permits delivery of fluids into distinct layers of a
vascular wall. The vascular wall consists of numerous structures
and layers, structures and layers, including the endothelial layer
and the basement membrane layer (collectively the intimal layer),
the internal elastic lamina, the medial layer, and the adventitial
layer. These layers are arranged such that the endothelium is
exposed to the lumen of the vessel and the basement membrane, the
intima, the internal elastic lamina, the media, and the adventitia
are each successively layered over the endothelium as described in
U.S. Pat. App. Publication No. 2006/0189941A1. With the medical
devices of the present invention, the depth to which the tissue
penetrator tips 156, 158 can penetrate into the target tissue can
be controlled by rotating the controllers 162, 164. For example, if
the target layer is the adventitial layer, the constrained energy
of the tubes 114, 116 is released, the tubes adopt their
unconstrained, curved configurations shown in FIG. 28, and the
tissue penetrator tips 156, 158 are advanced with the controllers
to a length sufficient for penetration to the depth of the
adventitial layer. Likewise, if the target layer is the medial
layer, the constrained energy of the tubes 114, 116 is released,
the tubes adopt their unconstrained, curved configurations shown in
FIG. 28, and the tissue penetrator tips 156, 158 are advanced with
the controllers to a length sufficient for penetration to the depth
of the medial layer.
[0415] With the tissue penetrators embedded in the desired layer of
the wall of the biological conduit, a fluid can then be delivered
through the tissue penetrators 118, 120. When the delivery of the
fluid is complete, the controllers 162, 164 can be operated to
withdraw the tissue penetrator tips 156, 158 back into the interior
bores 152, 154 of the tissue penetrator presentation tubes 114,
116. The deployment tube 122 can then be moved to its forward
position where the deployment tube distal end 172 moves the tissue
penetrator presentation tubes 114, 116 back to their constrained
positions shown in FIG. 27. When the deployment tube 122 has been
moved to its full forward position shown in FIG. 27, the assembly
can then be repositioned or withdrawn from the body.
[0416] The medical device of the instant invention also permits
delivery of fluids to plaque deposits on the inside of the wall of
the biological conduit or within the wall of the biological
conduit.
[0417] The medical device of the instant invention also permits
delivery of fluids to extracellular spaces or tissues located
outside of the outer wall of the biological conduit (e.g., to the
exterior surface of a blood vessel or to muscle positioned against
the outer surface of vessel such as myocardium).
[0418] One advantageous feature of the devices of the present
invention is that the actuators, by virtue of their design, make
contact with less than the complete circumference of the inner wall
of a biological conduit following their deployment therein. In
preferred embodiments, the actuators make contact with less than
100% of the circumference of the inner wall of a biological conduit
in which they are deployed. More preferably, the actuators make
contact with less than 75%, 50% or 25% of the circumference of the
inner wall of a biological conduit in which they are deployed. Most
preferably, the actuators make contact with less than 10%, 5%,
2.5%, 1%, 0.5% or 0.1% of the circumference of the inner wall of a
biological conduit in which they are deployed.
[0419] The devices can be used to deliver fluids comprising a
variety of therapeutic and/or diagnostic agents to a wall of a
biological conduit. Therapeutic agents include, but are not limited
to proteins, chemicals, small molecules, cells and nucleic acids. A
therapeutic agent delivered by the device may either comprise a
microparticle or a nanoparticle, be complexed with a microparticle
or a nanoparticle, or be bound to a microparticle or a
nanoparticle. Protein agents include elastases, antiproliferative
agents, and agents that inhibit vasospasm. The use of the devices
for delivery of an elastase is specifically contemplated. Several
published patent applications (WO 2001/21574; WO 2004/073504; and
WO 2006/036804) teach that elastase, alone and in combination with
other agents, is beneficial in the treatment of diseases of
biological conduits, including obstruction of biological conduits
and vasospasm. Diagnostic agents include, but are not limited to,
contrast, microparticles, nanoparticles or other imaging
agents.
[0420] A variety of distinct fluid delivery methods can be
practiced with the device. In certain applications, distinct fluids
can be delivered through each tissue penetrator of the device
either simultaneously or sequentially. In other applications, the
same fluid can be delivered through both tissue penetrators either
simultaneously or sequentially. Embodiments and/or methods where a
first fluid is delivered through both tissue penetrators followed
by delivery of a second fluid through both tissue penetrators are
also contemplated.
[0421] Methods of using the devices to deliver fluids into or
through a wall of a biological conduit are also specifically
contemplated. These methods comprise the steps of introducing the
device into the biological conduit, advancing the device to a
target site within the conduit, releasing the actuators from their
constrained positions, optionally advancing the tissue penetrators
through lumens in the actuators to penetrate to a desired depth
into the wall of a biological conduit, delivering at least one
fluid into or through the wall, optionally returning the tissue
penetrators back into the lumens of the actuators, retracting the
actuators to their constrained position, repositioning the device
in the same or a different conduit for the delivery of additional
fluid if so desired, and removing the device from the conduit.
6. EXAMPLES
[0422] This section describes methods of production of recombinant
type I elastase for clinical use, for example as an agent to
enlarge the diameter of blood vessels and thereby the lumen of
blood vessels. Human type I pancreatic elastase displays 89% amino
acid identity across the entire length of the porcine type I
pancreatic elastase, with complete conservation of the "catalytic
triad" and substrate specificity determining residues. Porcine type
I elastase is initially synthesized as an enzymatically inactive
proenzyme that is activated by trypsin to yield the mature enzyme
that contains four internal disulfide bonds and no glycosylation
(see Shotton, 1970, Methods Enzymol 19:113-140, Elastase; and
Hartley and Shotton, 1971, Biochem. J. 124(2): 289-299, Pancreatic
Elastase. The Enzymes 3:323-373 and references therein).
[0423] The examples below demonstrate the development of efficient
and scalable recombinant porcine and human type I elastase
expression and purification schemes suitable for cGMP manufacture
of these enzymes for non-clinical and clinical studies, and
commercial pharmaceutical use. The mature porcine elastase has been
given the name PRT-102. The mature human type I elastase has been
given the name PRT-201.
6.1 Terminology and Abbreviations
[0424] As used herein, the terms PRT-101, PRT-102, PRT-201 and
pro-PRT-201 shall mean the following:
[0425] PRT-101: porcine pancreatic elastase. Unless otherwise
indicated, the porcine pancreatic elastase employed in the examples
is highly purified porcine pancreatic elastase purchased from
Elastin Products Company, Inc, Owensville, Mo., catalog #EC134.
[0426] PRT-102: mature recombinant type I porcine pancreatic
elastase. It should be noted that vectors with the designation
"pPROT101-XXX" encode PRT-102.
[0427] PRT-201: mature recombinant type I human pancreatic
elastase.
[0428] pro-PRT-201: proenzyme form of recombinant human type I
pancreatic elastase containing a propeptide sequence.
[0429] The following abbreviations are used in Section 6 of the
application:
[0430] BKGY: buffered glycerol-complex medium
[0431] BKME: buffered methanol-complex medium
[0432] CHO: Chinese hamster ovary
[0433] E. coli: Escherichia coli
[0434] ELA-1: type I pancreatic elastase
[0435] HEK: human embryonic kidney
[0436] hELA-1: human ELA-1
[0437] HIC: hydrophobic interaction chromatography
[0438] MBP: maltose binding protein
[0439] pELA-1: porcine ELA-1
[0440] P. pastoris: Pichia pastoris
[0441] PCR: polymerase chain reaction
[0442] PMSF: phenylmethylsulphonyl fluoride
[0443] RP: reversed phase
[0444] S. cerevisiae: Saccharomyces cerevisiae
[0445] SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel
electrophoresis
[0446] SEC: size exclusion chromatography
[0447] USP: United States Pharmacopeia
[0448] YPDS: yeast extract peptone dextrose sorbitol medium
6.2 Example 1
Elastase DNA Synthesis
[0449] The human elastase-1 coding sequence was obtained from U.S.
Pat. No. 5,162,205 (Takiguichi et al, 1992). Several sequence
changes were made to facilitate cloning into expression vectors
(FIG. 1A). The base changes fell within the degeneracy of the
genetic code so that no amino acid residues were changed. A second
stop codon was added immediately after the native stop codon to
minimize potential ribosome read through.
[0450] The modified coding sequence was synthesized by Blue Heron
Biotechnology (Bothell, Wash.) using a non-PCR "long oligo"
technique under license from Amgen (Thousand Oaks, Calif.). The
recombinant DNA, named ELA-1.2A (SEQ ID NO: 81), was cloned into
the vector Blue Heron pUC, a derivative of pUC119, and the
resulting plasmid was named pPROT1. pPROT1 was sequenced on both
strands to confirm the correct sequence. High quality sequencing
data with extensive overlap on both strands permitted unambiguous
base assignments across the entire sequence, which was covered by a
minimum of four sequencing reactions with at least one reaction for
each strand.
6.3 Example 2
Expression of PRT-201 in E. coli
[0451] A variety of expression strategies were attempted in an
effort to obtain soluble and enzymatically active human elastase in
E. coli.
[0452] One set of E. coli expression vectors comprised in frame
fusions of human type I pancreatic elastase (ELA-1) to the carboxy
terminus of a Maltose Binding Protein (MBP) that was in turn fused
to an N-terminal secretory peptide. Both the human ELA-1 mature and
proenzyme coding sequences were cloned as in-frame C-terminal
fusions to the MBP coding sequence of plasmid pMAL-p2G (New England
Biolabs, Inc., Beverly, Mass.) to yield either pPROT3 (mature
ELA-1) or pPROT5 (ELA-1 proenzyme). Construction of pPROT3 was
effected by first obtaining by PCR mutagenesis a 6.6 kb mature
human ELA-1 encoding fragment with a SnaBI site at the N-terminal
valine codon of mature human ELA-1 and a HindIII site located 3' to
the mature ELA-1 termination codons. This PCR mutagenesis used a
pPROT1 template comprising the ELA-1.2A coding sequence (SEQ ID
NO:81) and two oligonucleotide primers (5' ATC TAC GTA GTC GGA GGG
ACT GAG GCC, SEQ ID NO:75; and 5' gtc gac aag ctt atc agt tgg agg
cga t, SEQ ID NO:76). The resultant 6.6 kb PCR fragment was
isolated, digested with SnaBI and HindIII, and subsequently cloned
into the XmaI/HindIII digested pMAL-p2G vector to yield pPROT3.
Construction of pPROT5 was effected by cloning the ScaI/HindIII
fragment from pPROT1 and ligating into the pMAL p2G vector that had
been digested with SnaBI and HindIII. The resultant fusion operably
links the N-terminus of the human ELA-1 proenzyme coding region to
the C-terminus of the MBP of pMAL-p2G in pPROT5. The trypsin
cleavage domain of the human ELA-1 proprotein is preserved in
pPROT5.
[0453] E. coli strain TB1 was transformed with pPROT3 and pPROT5
and subsequently induced with IPTG to determine if either the
fusion protein or enzymatically active human ELA-1 could be
produced. In the case of pPROT3, all of the MBP-ELA-1 fusion
protein produced was insoluble. No soluble or enzymatically active
MBP-ELA-1 fusion protein was detected in the periplasmic material
obtained by osmotic shock of induced pPROT3-containing E. coli. In
the case of pPROT5, low levels of soluble recombinant MBP-proELA-1
protein could be detected in the periplasmic material obtained by
osmotic shock of induced pPROT5-containing E. coli through use of
SDS-PAGE and Coomassie stain or by use of anti-MBP antibodies on a
Western blot (New England Biolabs, Inc., Beverly, Mass.). The
soluble recombinant MBP-proELA-1 protein could be digested with
trypsin to yield both MBP and mature human ELA-1. Mature human
ELA-1 obtained from inductions of pPROT5 was subsequently assayed
for elastase activity with SLAP peptide substrate. Elastase
activity was observed in pPROT5 periplasmic extracts. This elastase
enzymatic activity was dependent on trypsin activation (i.e., no
activity was observed in absence of trypsin and amount of activity
is increased by increasing the time period of trypsin activation).
Moreover, the elastase activity was dependent on pPROT5 in as much
as no activity was observed in pMAL-p2G vector control extracts
treated in parallel with trypsin. Finally, the pPROT5 elastase
activity was inhibited by PMSF (a known serine protease
inhibitor).
[0454] The recombinant MBP-proELA-1 fusion protein was subsequently
purified and cleaved with trypsin to obtain an enzymatically active
pPROT5-derived mature human ELA-1. The fusion protein was first
purified on amylose affinity chromatography followed by elution
with maltose. The purified MBP-proELA-1 was then treated with
immobilized trypsin. Following the trypsin activation step, the
cleaved MBP-proELA-1 was purified by cationic SP Sepharose
chromatography. However, subsequent experiments with affinity
purified pPROT5-derived mature human ELA-1 indicated that only very
limited amounts of soluble and enzymatically active elastase could
be obtained from E. coli containing pPROT5. Moreover, the specific
activity of the affinity purified, pPROT5-derived mature human
ELA-1 was very low, ranging from 0.27 to 0.38 U/mg (U=micromole of
SLAP substrate hydrolyzed per min).
[0455] An alternative strategy of obtaining soluble and
enzymatically active ELA-1 in E. coli was also pursued. In brief,
the pPROT8 vector that encodes a protein fusion comprising the
first 8 amino acid residues of the E. coli lacZ alpha subunit plus
5 amino acids of poly linker encoded residues followed by the human
ELA-1 N-terminal proenzyme was constructed. This vector was
constructed by ligating a BamHI/NcoI fragment containing the human
ELA-1 coding sequence from pPROT1 (i.e., a vector containing the
ELA-1.2A sequence; SEQ ID NO: 81) into pBlueHeron pUC (Blue Heron
Biotechnology, Bothell, Wash., USA) that was digested with
BamHI/NcoI.
[0456] E. coli strain EC100 (EPICENTRE Biotechnologies, Madison,
Wis.) was transformed with pPROT8 and subsequently induced with
IPTG to determine if either the fusion protein or enzymatically
active human ELA-1 could be produced. In the case of EC100
transformed cells, all of the pPROT8 derived LacZ-proELA-1 fusion
protein produced was insoluble (i.e., found in inclusion bodies).
An E. coli strain containing mutations in the trxB and gor genes
(the Origami.TM. strain, Takara Mirus Bio, Inc., Madison, Wis.) was
subsequently transformed with pPROT8 as E. coli strains with
mutations in these coding sequences are known to promote recovery
of soluble and enzymatically active recombinant proteins. Although
some soluble pPROT8 derived LacZ-proELA-1 fusion protein in the
trxB/gor E. coli strain was recovered upon induction with IPTG, it
could not be converted to enzymatically active human ELA-1 with
trypsin.
6.4 Example 3
Expression of PRT-201 in Mammalian Cell Lines
[0457] Several expression strategies were attempted in an effort to
obtain soluble and enzymatically active human type I pancreatic
elastase (ELA-1) in mammalian cell lines. The high copy mammalian
expression vector pcDNA3.1 (Invitrogen) containing the CMV promoter
was used as a backbone for two human ELA-1 elastase expression
vectors, pPROT30 and pPROT31. To construct pPROT30, the human ELA-1
proenzyme sequence was amplified by PCR and fused to a porcine
pancreatic elastase signal sequence incorporated in the forward PCR
primer. Using restriction sites incorporated into the PCR primers,
the PCR product was digested and ligated using the corresponding
restriction sites in the pcDNA3.1 vector. pPROT31 was constructed
in a similar fashion, except that the human ELA-1 mature coding
sequence was used instead of the proenzyme coding sequence in an
attempt at direct expression of the mature enzyme. E. coli was
transformed with the ligation reactions and clones were selected
for miniprep screening. One clone for each expression vector was
selected based on expected restriction digest patterns for the
correct insert. Plasmid DNA was prepared for each clone and the
expression vector coding sequences were confirmed by DNA
sequencing.
[0458] The mammalian cell lines CHO, COS, HEK293 and HEK293T were
transiently transfected separately with pPROT30 and pPROT31. After
several days, cell culture supernatants were harvested and analyzed
for human ELA-1 proprotein (pPROT30) or mature protein (pPROT31)
expression by Western blot. An anti-porcine pancreatic elastase
polyclonal antibody cross-reacted with a band of the expected
molecular weight for the human ELA-1 proprotein in pPROT30
supernatants and for the mature human ELA-1 protein in pPROT31
supernatants.
[0459] The pPROT30 and pPROT31 supernatants were analyzed for
elastase activity by SLAP assay. For pPROT30, the supernatants were
first treated with trypsin to convert the proenzyme to mature
PRT-201. No elastase activity was detected in any of the
supernatants for either vector using the SLAP assay.
6.5 Example 4
Expression of Trypsin-Activated PRT-201 in P. pastoris
[0460] The vector for P. pastoris secreted expression, PV-1, was
synthesized by Blue Heron and used to first clone the wild-type
human ELA-1 coding sequence. The PV-1 vector was designed for
simple cloning, selection and high-level expression of the
recombinant protein. The vector contains the Zeocin.TM. resistance
gene for direct selection of multi-copy integrants. Fusion of the
N-terminus of the elastase propeptide to a yeast .alpha.-mating
type sequence comprising the yeast secretion signal, propeptide and
spacer sequences as shown in FIG. 1B permits secretion of the
expressed protein in the culture media. The secreted elastase
proprotein can be easily separated from the cell pellet, a
substantial first step towards purification. Additionally, the
proenzyme form of human ELA-1 containing the trypsin cleavage site
was selected for expression to avoid directly expressing the
mature, activated enzyme which may lead to protein misfolding or
toxicity to the cells expressing the recombinant enzyme.
[0461] Cloning of the expression construct pPROT24-V to direct the
expression of ELA-1 proenzyme was accomplished as follows. The
human ELA-1 coding region (SEQ ID NO:81) was amplified from Blue
Heron pUC ELA-1 by PCR (Expand High Fidelity PCR System, Roche,
Indianapolis, Ind.). The 20F forward primer incorporated an XhoI
site
(5'-ggctcgagaaaagagaggctgaagctactcaggaccttccggaaaccaatgcccgg-3; SEQ
ID NO:35). The 24R primer incorporated a SacII site
(5'-gggccgcggcttatcagttggaggcgatgacat-3'; SEQ ID NO:36). The
resulting PCR product was gel-purified and cloned into pCR2.1-TOPO
(Invitrogen). The ELA-1 coding sequence was isolated using XhoI and
SacII, gel-purified, and cloned into PV-1 vector at those sites to
yield pPROT24-V (FIG. 3).
[0462] The pPROT24-V ligated product was amplified in E. coli
strain TOP10. The cell mixture was plated on low salt LB plates
supplemented with 25 microgram/mL Zeocin. DNA plasmid was prepared
(Qiagen, Valencia, Calif.) and the human ELA-1 insert was
identified by restriction digestion. The pPROT24 coding sequence
was verified by sequencing both strands of purified maxiprep DNA
with multiple overlapping reactions. High quality sequencing data
allowed unambiguous base assignments and confirmed the correct
coding sequence. Glycerol stocks of pPROT24-V/TOP10 were made and
stored at -80.degree. C.
[0463] The wild-type NRRL Y-11430 P. pastoris strain obtained from
the United States Department of Agriculture (USDA, Peoria, Ill.,
USA) was used for transformation. Plasmid DNA from pPROT24-V was
linearized with SacI and complete digestion was confirmed by
running a small aliquot of the reaction on an agarose gel.
Electroporation was used to transform P. pastoris with pPROT24-V
plasmid DNA. Cell mixtures were plated onto YPDS plates containing
100 microgram/mL Zeocin. After three days, colonies began to form
and were selected over several more days for re-streaking on fresh
plates.
[0464] In general, drug-resistant transformants were screened for
expression in a 1 L baffled flask. A single colony was used to
inoculate 200 mL of BKGY medium. The composition of the BKGY
solution was the following: 10 g/L glycerol, 13.4 g/L yeast
nitrogen base with ammonium sulfate and without amino acids
(Invitrogen), 20 g/L soy peptone, 10 g/L yeast extract, 0.4 mg/L
biotin in 0.1 M potassium-phosphate buffer (pH 5.0). The culture
was grown for two days at 28.degree. C. with shaking at 275 rpm.
The cultures were pelleted by centrifugation at 650.times.g for 10
min at room temperature. The cell pellets were resuspended with
BKME induction media, pH 5.0, by resuspending the pellets at a
ratio of 1 g wet cells to 5 mL induction media. A 50 mL cell
suspension was placed in a 500 mL non-baffled flask to obtain a
1:10 ratio of cell suspension to flask volume. The cells were
incubated at 22.degree. C. with shaking at 275 rpm for 1-3 days.
Methanol in the induction media was replenished to a final
concentration of 0.5% twice daily over the course of induction.
[0465] To screen for expression, 1 mL aliquots were taken,
transferred to 1.5 mL microfuge tubes and centrifuged for 5 min at
20,000.times.g in a microcentrifuge. Supernatants were transferred
to fresh tubes and stored at -80.degree. C. For SDS-PAGE analysis,
supernatant aliquots were thawed and mixed with 4.times. Laemmli
buffer supplemented to 5% volume with the reducing agent
beta-mercaptoethanol. Samples were boiled for 5 min, gently
centrifuged, and loaded onto an 8-16% gradient Tris-HCl pre-cast
gel in a Criterion electrophoresis system (Bio-Rad). After
electrophoresis, the gel was stained with Coomassie and analyzed
for human ELA-1 proenzyme expression. Clone 201-24-266-VU was
selected as a high-yield clone for further evaluation. A
development cell bank consisting of 201-24-266-VU glycerol stocks
was prepared and stored at -80.degree. C.
[0466] For scale-up production of human ELA-1 proenzyme using clone
201-24-266-VU, multiple production runs were performed that
generally followed the methods described below. Where applicable,
run-to-run variations in the methods are noted.
[0467] For cell culture, a series of 2 L baffled shaker flasks
(typically ranging from 20 to 40 flasks) containing 500 mL BKGY
growth medium were inoculated with 250 microliters of thawed
201-24-266-VU glycerol stock. Cultures were grown at 28.degree. C.
for 2 days in a shaking incubator at 250-300 rpm. After 2 days, the
cells were pelleted by centrifugation and the supernatant was
discarded. The cells were resuspended in BKME induction media, pH
5.0, at a ratio of 1 g of weight cells to 5 mL media. A volume of
200-400 mL cell suspension was placed in 2 L non-baffled flasks and
cultured for 3 days in a shaking incubator (250-300 rpm) at
22.degree. C. Methanol in the media was replenished to 0.5% volume
twice daily during the course of induction. At the end of
induction, the shake flask cultures were centrifuged to pellet the
cells. The supernatant was removed and immediately filtered at room
temperature through a 0.22 um polyethersulfone membrane using 1 L
vacuum filtration units to remove any remaining cell debris. The
filtrate was stored at 2-8.degree. C. for up to 1.5 months. Based
on HIC-HPLC analysis, the yield of human ELA-1 proenzyme from clone
pro-PRT-201-24-266-VU in the clarified supernatant was typically
200 to 250 mg/L.
[0468] Capture of pro-PRT-201-24-266-VU from the supernatant was
effected as follows. First, supernatant from multiple rounds of
shaker flask cultures (typically 4 to 10 rounds) was combined
(typically 8 to 25 L total), diluted 8-fold with water and adjusted
to pH 5.0 with 1 M HCl. The diluted supernatant was then loaded
onto an equilibrated 2 L bed volume Macro-Prep High S ionic
exchange capture column at 2-8.degree. C. at a rate of 100 mL/min
(linear flow rate 76 cm/hr). The chromatography program comprised
the following steps: 1. Wash column with 10 L (5 column volumes
[CVs]) of Buffer A (20 mM sodium citrate, pH 5.0.) at 100 mL/min
(76 cm/hr); 2. Wash column with 4 L (2 CVs) of a mixture of 90%
Buffer A and 10% Buffer B (500 mM sodium chloride; 20 mM sodium
citrate, pH 5.0) for a final buffer composition of 50 mM sodium
chloride; 20 mM sodium citrate, pH 5.0 at 100 mL/min (76 cm/hr); 3.
Wash column with 6 L (3 CVs) of a mixture of 80% Buffer A and 20%
Buffer B for a final buffer composition of 100 mM sodium chloride;
20 mM sodium citrate, pH 5.0 at 100 ml/min (76 cm/hr); 4. Wash
column with 6 L (3 CVs) of a linear gradient, starting from 75%
Buffer A and 25% Buffer B to 68% Buffer A and 32% Buffer B, at 100
ml/min (153 cm/hr); 5. Elute with a linear gradient of 30 L (15
CVs) starting from 68% Buffer A and 32% Buffer B to 0% Buffer A and
100% Buffer B, at 100 ml/min (76 cm/hr). The eluate was collected
in fractions of 500-1000 mL each.
[0469] Typically, two predominant protein species were observed by
SDS-PAGE followed by Coomassie staining: the glycosylated human
ELA-1 proenzyme and the non-glycosylated human ELA-1 proenzyme, as
determined by subsequent LC/MS analysis, typically eluting at about
320 mM sodium chloride, shown in FIG. 4. In some production runs a
protein slightly smaller than the human ELA-1 proenzyme was
observed as a minor species. Subsequent LC/MS analysis of these
fractions showed that most of the protein was not full-length
proenzyme but instead was lacking several amino acids at the
N-terminus. These N-terminal variants were purified and subjected
to elastase activity analysis, which revealed that they had lower
elastase activity than full length PRT-201. N-terminal variants
could arise from the human ELA-1 proenzyme exhibiting a low level
of elastase activity during cell culture and capture chromatography
operations and either cleaving itself through an intramolecular
reaction or cleaving another human ELA-1 proenzyme molecule through
an intermolecular reaction. Suboptimal cleavage conditions during
these operations could lead to a high level of inaccurate cleavage
of the proenzyme (sometimes referred to as spontaneous or
uncontrolled conversion) resulting in mostly N-terminal variants,
rather than intact, full length PRT-201.
[0470] After inspection of SDS-PAGE results, a subset of fractions
was pooled to obtain purified non-glycosylated pro-PRT-201 for
further processing. Fractions containing glycosylated proenzyme or
full-length PRT-201 and/or N-terminal variants (which co-migrate on
SDS-PAGE) were typically excluded from the pooling. The pooled
pro-PRT-201 was typically stored in a 5 L plastic beaker at
2-8.degree. C. for several hours to overnight prior to conversion
from proenzyme to mature enzyme.
[0471] Conversion of pro-PRT-201 to mature PRT-201 by immobilized
trypsin was effected as follows. The pooled pro-PRT-201 fractions
were dialyzed in 20 mM sodium phosphate, pH 5.0, overnight at
2-8.degree. C. This step provides for the removal of citrate, which
inhibits trypsin. Following dialysis, the pro-PRT-201 was passed
over a column of recombinant trypsin (TrypZean) immobilized to
agarose beads pre-equilibrated with 20 mM sodium phosphate, pH 5.0.
Typically, the contact time between pro-PRT-201 and the immobilized
TrypZean was between 3.5 to 5 min. The resulting post-conversion
material was analyzed by SDS-PAGE to confirm conversion and
subsequently loaded onto a Macro-Prep High S polish column. The
column was washed sequentially with 5 CVs of 20 mM sodium citrate,
pH 5.0; 2 CVs of 20 mM sodium citrate, 50 mM sodium chloride, pH
5.0; and 3 CVs of 20 mM sodium citrate, 100 mM sodium chloride, pH
5.0. The column was further washed with a linear gradient from 125
mM sodium chloride to 160 mM sodium chloride in 20 mM sodium
citrate, pH 5.0. PRT-201 was eluted in a linear gradient from 165
mM to 500 mM sodium chloride in 20 mM sodium citrate, pH 5.0 and
collected in fractions. Fractions were analyzed for protein by
SDS-PAGE followed by Coomassie staining and for elastase activity
by SLAP assay. Typically, fractions having a specific activity of
90% or greater than that of the fraction with the highest specific
activity were pooled. In subsequent LC/MS analysis, late-eluting
fractions having lower specific activity were found to be enriched
in PRT-201 N-terminal variants.
[0472] Pooled fractions with high specific activity were
diafiltered into formulation buffer composed of 0.1.times.PBS (13.7
mM sodium chloride, 1 mM sodium phosphate, 0.27 mM potassium
phosphate) at pH 5.0. After concentration of PRT-201 to 1 mg/mL,
the pH was adjusted to 7.4. The solution was then aliquoted into
glass serum vials with an elastomer stopper, and lyophilized.
Lyophilization was typically performed with a primary drying cycle
at -30 to -50.degree. C. and a secondary drying cycle at
-15.degree. C. After lyophilization, the vials were stoppered under
vacuum and crimped with an aluminum seal. The vials were then
typically stored at 2-8.degree. C. or -80.degree. C.
[0473] To evaluate the stability of lyophilized PRT-201 in vials,
two lots that were manufactured into sterile GMP drug product have
been placed on a stability program. The stability program consists
of storage of the drug product at -15.degree. C. and periodic
removal of a subset of vials for testing by the following
stability-indicating analytical methods (specifications are in
parentheses): appearance of lyophilized material (white to
off-white powder), appearance after reconstitution (clear,
colorless solution free of particles), specific activity by SLAP
assay (25-45 U/mg), purity by RP-HPLC (total purity: not less than
93%; individual impurity: not more than 2%), purity by reduced
SDS-PAGE (not less than 93%), purity by non-reduced SDS-PAGE (not
less than 93%), particulate matter injections (conforms to USP),
aggregates by SEC-HPLC (not more than 3%), content per vial
(4.5-5.5 mg), pH (6.5-8.5), moisture (does not exceed 5%) and
sterility (conforms to USP). To date, both lots have met the
indicated specifications at all time points tested (lot C0807117
through 12 months and lot C1007132 through 9 months). These results
indicate that PRT-201 is stable for at least 12 months when stored
lyophilized in vials at -15.degree. C.
[0474] The use of sodium citrate, pH 5.0, during both capture
chromatography of pro-PRT-201 and polish chromatography of
converted PRT-201 was based on data showing that sodium citrate
inhibited elastase activity of purified PRT-201 and therefore might
also inhibit elastase activity of pro-PRT-201 and PRT-201 during
processing operations. Such inhibition could minimize the
spontaneous conversion of pro-PRT-201 to N-terminal variants and
minimize auto-degradation of PRT-201. In an experiment where SLAP
assay buffer contained 115 mM sodium citrate, pH 5.0, the specific
activity of PRT-201 was inhibited by 91%.
[0475] Occasionally, eluted material from the pro-PRT-201 capture
column was not subjected to conversion by trypsin as described
above but instead used to obtain preparations enriched in various
protein species. For example, pools of fractions containing
primarily glycosylated pro-PRT-201, non-glycosylated pro-PRT-201 or
proteins arising from spontaneous conversion were made from the
capture column eluate. Typically, these fraction pools were
diafiltered into 10 mM sodium phosphate, pH 5.0, lyophilized, and
stored at -80.degree. C.
[0476] A study was performed to determine the effect of pH and
temperature on the stability of purified pro-PRT-201 over time.
Lyophilized pro-PRT-201 was reconstituted in 10 mM sodium phosphate
at pHs ranging from 3.0 to 8.4, followed by incubation at 4.degree.
C. or 25.degree. C. for 7 days. After 7 days, the pro-PRT-201
samples under conditions of pH 4.0-8.0 and 25.degree. C. showed an
enrichment in mature PRT-201 that increased with increasing pH as
shown by SDS-PAGE and Coomassie staining. Under the conditions of
pH 8.4 and 25.degree. C., complete conversion of pro-PRT-201 to
mature PRT-201 was observed. The samples at 4.degree. C., from pH
4.0 to 8.4 showed little to no conversion. At pH 3.0, no conversion
was observed at either 4.degree. C. and 25.degree. C. It has been
reported in the literature (Hartley and Shotton, 1971, Pancreatic
Elastase. Enzymes 3:323-373) that porcine pancreatic elastase is
irreversibly inactived after prolonged storage at pHs lower than
3.0. Thus, based these results of this study and to avoid
irreversibly inactivating PRT-201, useful conditions to minimize
conversion of purified pro-PRT-201 are storage at 4.degree. C.
between pH 3.0-4.0.
6.6 Example 5
Expression of Auto-Activated PRT-201 in P. pastoris
[0477] To obtain a variant proenzyme capable of auto-activation,
thereby eliminating the need for trypsin activation, a variety of
elastase cleavage domain variant vectors were constructed and
analyzed in small-scale culture and conversion experiments. The
variant vectors were created by site-directed PCR mutagenesis of
the pPROT24-V vector and subsequent derivative vectors.
Site-directed mutagenesis was performed using Pfu Turbo DNA
polymerase (Stratagene). The E. coli XL10-Gold strain was
transformed with the resulting plasmids. The transformed cell
mixtures were plated on low salt LB plates supplemented with 25
microgram/mL Zeocin. Drug-resistant clones were picked and plasmid
DNA was prepared (Qiagen, Valencia, Calif.). Clones were confirmed
for the expected codon changes by sequencing both strands of
plasmid DNA in the propeptide region with multiple overlapping
reactions. P. pastoris was transformed with the variant vectors and
clones were selected as described in the preceding Example.
[0478] A summary of the elastase cleavage domain variants that were
created is provided in Table 4 below:
TABLE-US-00004 TABLE 4 Elastase cleavage domain variants
Pro-peptide sequence Cleaved bond name P5 P4 P3 P2 P1 P'1 P'2 P'3
24 Glu Thr Asn Ala Arg Val Val Gly 40 Glu Thr Ala Ala Ala Val Val
Gly 41 Glu Thr Asn Ala Ala Ala Val Gly 42 Glu Thr Asn Ala Ala Val
Val Gly 43 Glu Thr Asn Ala Pro Val Val Gly 44 Glu Thr Gly Ala Gly
Ile Val Gly 45 Glu Thr Val Pro Gly Val Val Gly 46 Glu Thr Ala Pro
Gly Val Val Gly 47 Glu Thr Asn Pro Gly Val Val Gly 48 Glu Thr Asn
Pro Ala Val Val Gly 49 Glu Thr Asn His Ala Val Val Gly 52 Glu Thr
Lys Pro Ala Val Val Gly 53 Glu Thr His Pro Ala Val Val Gly 54 Glu
His Asn Pro Ala Val Val Gly 55 His Thr Asn Pro Ala Val Val Gly 56
Pro Thr His Pro Ala Val Val Gly 57 Pro Thr Asn Pro Ala Val Val Gly
58 His Thr His Pro Ala Val Val Gly 59 Glu Thr Phe Pro Ala Val Val
Gly 60 His Thr Phe Pro Ala Val Val Gly 61 Gly Thr Phe Pro Ala Val
Val Gly 62 His Thr Gly Pro Ala Val Val Gly 63 His Thr Lys Pro Ala
Val Val Gly
[0479] For Table 4 above, the first column listing of the
"Pro-Peptide Sequence Name" corresponds to the SEQ ID NO for the
indicated elastase cleavage domain. Thus, 24 corresponds to the
wild-type trypsin cleavage domain of SEQ ID NO:24. Numbers 40-49,
52-63 correspond respectively to the variant elastase cleavage
domains of SEQ ID NOS: 40-49, and 52-63.
[0480] To culture the variant clones, shaker flask culture
conditions developed for the trypsin-activated 201-24-266-VU clone
described in the preceding Example were generally followed. Several
methods of converting the variant proproteins secreted into the
shaker flask supernatant to mature PRT-201 were tested. The first
conversion strategy consisted of chromatographically purifying the
variant proenzyme first, followed by controlled cleavage in a
specific conversion buffer. Because the amino acid changes in the
variant proproteins resulted in only small changes in the
theoretical isoelectric points compared to the wild-type proenzyme,
cation exchange chromatography was carried out generally as
described in the preceding Example. Supernatants from the variant
clone cultures were prepared for chromatography either by dilution
with water generally as described in the preceding Example or by
concentration followed by diafiltration of the supernatant using
tangential flow filtration into the column loading buffer. After
chromatographic purification, eluted fractions were analyzed by
SDS-PAGE followed by Coomassie staining. Gel analysis demonstrated
greater amounts of converted mature protein to proprotein in the
fractions compared to the starting supernatant, indicating that a
considerable amount of spontaneous proprotein conversion had
occurred. Upon subsequent purification, the spontaneously converted
protein was determined by LC/MS to consist of mainly N-terminal
variants which were shown to have little or no elastase activity in
the SLAP assay.
[0481] The second conversion strategy consisted of converting the
variant proprotein prior to purifying from the culture supernatant,
followed by chromatographic purification of the mature enzyme. This
conversion strategy was first tested in a small-scale assay and
subsequently scaled up to accommodate larger conversion volumes.
For small-scale conversion, clarified supernatant from variant
clone cultures was typically concentrated 5-fold by centrifugation
at 2-8.degree. C. in an ultracentrifugal filter device. After
concentration, the retentate was diluted 5-fold with Tris buffer to
a final concentration of 100 mM of Tris-HCl typically in a pH range
of 8.0 to 9.0. Samples were incubated at room temperature on a
rocking platform. Elastase activity was monitored by SLAP assay
typically until the activity reaction velocity reached a plateau.
In some cases, the reaction velocity increased so slowly that SLAP
monitoring was halted before a plateau was achieved. Converted
samples were analyzed for protein species (e.g., proprotein and
PRT-201/N-terminal variants) by SDS-PAGE. To scale up this
conversion strategy, supernatant containing variant proprotein was
concentrated 10-fold using tangential flow filtration followed by
diafiltration with 100 mM Tris-HCl ranging in pH from 6.0 to 9.0.
The progress of the conversion reaction was monitored over time by
HIC-HPLC analysis in which proprotein, PRT-201, and N-terminal
variant species were quantified. When the pH of the diafiltration
buffer was between 8.0 and 9.0 there were generally higher rates of
conversion.
[0482] The elastase proproteins listed in Table 5 were expressed in
P. pastoris as described and tested for their capacity to undergo
auto-conversion in small-scale conversion assays as described in
the second conversion strategy above. The results of those studies
are summarized in Table 5 below:
TABLE-US-00005 TABLE 5 Results of expression of elastase
proproteins in P. pastoris. Pro-Peptide Shaker Flask Shaker Flask %
N-Terminal Trypsin Used in Sequence Name Yield Stability Conversion
Rate Variants Processing 24 High High Fast 20% Yes 40 None Not
Applicable Not Applicable Not Applicable No 41 Intermediate High
Slow Not Tested No 42 Low Low Intermediate 25% No 43 Intermediate
High Slow Not Tested No 44 Intermediate-High High No Conversion Not
Applicable No Detected 45 Intermediate High No Conversion Not
Applicable No Detected 46 Intermediate High No Conversion Not
Applicable No Detected 47 Intermediate High No Conversion Not
Applicable No Detected 48 Intermediate Low Fast 15% No 49 High High
Slow 35% No 52 Intermediate Low Fast Not Tested No 53 Intermediate
Intermediate Fast 25% No 54 Intermediate Low Fast Not Tested No 55
Intermediate - Intermediate Fast 15% No High 56 Low Low Not Tested
Not Tested No 57 Low Low Not Tested Not Tested No 58 Intermediate -
Intermediate Slow Not Tested No High 59 Intermediate - Intermediate
Slow Not Tested No High 60 Intermediate - High Slow Not Tested No
High 61 None Not Applicable Not Applicable Not Applicable No 62
High High No Conversion Not Applicable No Detected 63 None Not
Applicable Not Applicable Not Applicable No
[0483] In Table 5 above, the first column listing of the
"Pro-Peptide Sequence Name" corresponds to the SEQ ID NO for the
indicated elastase cleavage domain. Thus, 24 corresponds to the
wild-type trypsin activated elastase cleavage domain of SEQ ID
NO:24. Numbers 40-49, and 52-63 correspond respectively to the
variant elastase cleavage domains of SEQ ID NOS: 40-49, and 52-63.
The column labeled "Shaker Flask Yield" corresponds to the amount
of the corresponding proprotein in the culture supernatant over 3
days of induction as determined by SDS-PAGE analysis. The column
labeled "Shaker Flask Stability" corresponds to the stability of
the corresponding proprotein in the supernatant of the shaker flask
culture media over 3 days of induction as determined by the amount
of PRT-201/N-terminal variants seen on SDS-PAGE analysis. The
column labeled "Conversion Rate" corresponds to the relative rate
of conversion of proprotein to PRT-201, as indicated by the time to
achieve maximal SLAP reaction velocity (Fast: less than 60 minutes;
Intermediate: 60 to 120 minutes; and Slow: greater than 120
minutes). Conversion time courses of the variant proproteins were
determined using the small-scale conversion assay described above
and compared to the conversion time course of the 24 proprotein
determined by activation using immobilized trypsin. The column
labeled "% N-Terminal Variants" refers to the percentage of
converted protein that comprised N-terminal variants of the mature
elastase protein (i.e. variants comprising cleavage at the bond
C-terminal to any site other than P1). To illustrate the relative
ranking systems used in in Table 5, examples of SDS-PAGE,
conversion rate and N-terminal variant analyses for a subset of
auto-activated variants are shown in FIG. 5.
[0484] Analysis of the various variants revealed that elastase
proproteins comprising either the SEQ ID NO:48 or SEQ ID NO:55
variant elastase cleavage domain provided auto-activated elastases
with superior qualities including intermediate to high shaker flask
yields and low percentages of variants upon conversion. Further
analysis of elastase proproteins comprising either the SEQ ID NO:48
and SEQ ID NO:55 variant elastase cleavage domain revealed that
auto-activation of the corresponding proproteins (i.e. the elastase
proenzymes of SEQ ID NO:64 and SEQ ID NO:69, respectively) produced
just one class of N-terminal variant with a cleavage at the peptide
bond C-terminal to the P'2 residue. Further analysis of elastase
proproteins revealed that the proprotein comprising SEQ ID NO:55
variant elastase cleavage domain was more stablethan the proprotein
comprising the SEQ ID NO:48 variant elastase cleavage domain.
[0485] Initial experiments to optimize conditions for controlled
cleavage of purified pro-PRT-201 were performed using the
proprotein with the 42 pro-peptide sequence (SEQ ID NO:6). This
purified proprotein was subjected to conversion in a matrix of
conditions including pH (7.7 to 8.9), buffer composition (0.4 to 10
mM sodium citrate), protein concentration (0.14 to 0.23 mg/mL), and
reaction time (5 to 24 hours). At the end of the conversion period,
the reactions were quenched by adding formic acid to reduce the pH
to 3.0. The relative amounts of protein species in each reaction
were determined by mass spectrometry. Based on these results, the
conversion conditions that resulted in the lowest percentage of
N-terminal variants included a pH of 8.3, a buffer composition of
100 mM Tris and less than 1 mM sodium citrate, a protein
concentration of 0.2 mg/mL, and a reaction time of 5 to 24 hours.
In this study, only the reaction end points were analyzed. Thus
real-time data on conversion quality was not obtained, and the
final result may have reflected both the initial production of
N-terminal variants and a substantial amount of time for those
variants to have been degraded. Subsequently, an HIC-HPLC assay was
developed to enable real-time monitoring of the conversion
reaction. Further conversion optimization studies, including those
using real-time HIC-HPLC monitoring, are described in Example
6.
6.7 Example 6
Expression of Auto-Activated PRT-201 in P. pastoris Using a
Multicopy Variant Vector
[0486] Multicopy integration of recombinant genes in P. pastoris
has been utilized to increase expression of the desired protein
(see, e.g., Sreekrishna et al., 1989, Biochemistry 28:4117-4125;
Clare et al., 1991, Bio/Technology 9:455-460; Romanos et al., 1991,
Vaccine 9:901-906). However, in certain instances, expression
levels obtained from single copy vector integrants was efficient
and was not improved by the multicopy vector integrants (Cregg et
al., 1987, Bio/Technology 5:479-485). Spontaneous multicopy plasmid
integration events occur in vivo at a low frequency in P. pastoris.
To obtain genomic integration of multiple copies of a gene and
possibly increase the protein expression, an in vitro ligation
method can be used to produce tandem inserts of the gene in an
expression vector, followed by P. pastoris transformation.
[0487] To obtain a multicopy integrant of the pPROT55-V variant, an
in vitro ligation method was used to construct a vector containing
multiple copies of the pPROT55-V that was subsequently used for P.
pastoris transformation. To make the multicopy vector, the
pPROT55-V vector was digested with BglII and BamHI to release the
2.3 kb expression cassette encoding the pro-PRT-201 gene, the AOX1
promoter, and the AOX1 transcription termination sequence. The
expression cassette was then ligated with a preparation of the
pPROT55-V vector that had been linearized with BamHI and treated
with calf intestinal alkaline phosphatase (New England Biolabs,
Mass., USA) to prevent self-ligating. The ligation mixture was
incubated overnight at 16.degree. C. The E. coli TOP 10 strain
(Invitrogen, Calif., USA) was transformed with the ligation
reaction. The transformation mix was plated out on low salt LB in
the presence of 25 microgram/mL of Zeocin. Drug-resistant
transformants were picked and plasmid DNA was prepared.
[0488] To determine the number of expression cassettes in the
resulting clones, plasmid DNA was digested with BglII and BamHI and
analyzed by agarose gel electrophoresis with a DNA size standard
marker. A clone containing a single defined insert band with the
size consistent with three 2.3 kb expression cassettes was
identified and named pPROT55M3-V. Restriction enzyme mapping was
used to confirm the orientation of a linear head-to-tail multimer
formation for the pPROT55M3-V vector. FIG. 6 depicts the
pPROT55M3-V cloning scheme.
[0489] The wild-type P. pastoris strain NRRL Y-11430 was used for
transformation, which was carried out as described in Example 4
except that the pPROT55M3-V vector was linearized with BglII
instead of SacI prior to transformation. Drug-resistant
transformants were cultured and screened for expression of the
pPROT55M3 proprotein as described in Example 4.
[0490] Optimization of shaker flask culture conditions was
performed to minimize spontaneous cleavage during induction. Shaker
flask optimization focused on two variables, induction temperature
and induction media composition. First, it was found that
performing induction at 22.degree. C. compared to 25.degree. C.
resulted in a higher ratio of proenzyme to mature enzyme in the
culture supernatant for all media compositions tested. Second,
addition of sodium citrate to increase the buffering strength of
the induction media resulted in the absence of spontaneously
converted mature enzyme in the culture supernatant across all
sodium citrate concentrations tested (12.5 to 50 mM). The effects
of these variables on proprotein expression yield and stability in
shaker flask supernatant over time are illustrated in FIG. 7.
[0491] The high-expressing three-copy clone 201-55M3-003-VU was
selected for scale-up fermentation analysis using fermentation
procedures established for the 201-55-001-VU clone described as
follows. Fermentation of the 201-55-001-VU clone was effected by
thawing one cell bank vial and using it to inoculate a shaker flask
containing 500 ml of BKGY growth medium at pH 5.7. The seed culture
was grown for 24 hours with shaking at 28.degree. C. until the wet
cell weight was approximately 40 g/L. The fermentor containing BKGY
growth medium at pH 5.7 was sterilized in the autoclave. After the
media was cooled to 28.degree. C., supplements including yeast
nitrogen base and biotin were added. The fermentor was inoculated
at a ratio of 1:33 of seed culture to BKGY growth medium.
[0492] The fermentation procedure started with a fed-batch of
glycerol and glycerol feed at pH 5.7 at 28.degree. C. The pH was
controlled by 10% phosphoric acid and 30% ammonium sulfate
solutions. The culture was agitated from 300-1000 rpm with aeration
to control the dissolved oxygen at 40%. After the initial glycerol
batch was depleted and dissolved oxygen spiked, indicating the
depletion of glycerol from the system, additional 50% glycerol was
fed at 131 g/h until the wet cell weight reached preferably between
200 g/L to 300 g/L. After the wet cell weight reached 200 g/L-300
g/L, the induction was immediately initiated with a methanol bolus
of 0.025 mL per gram of wet biomass. After depletion of the
methanol bolus and the rise of dissolved oxygen, induction was
continued by the addition of limiting amounts of methanol with a
constant feed rate of 0.0034 g methanol/g wet cell weight/hr. At
the start of constant methanol feed, the pH of the fermentation
broth was changed from 5.7 to 5.5 and the temperature was changed
from 28.degree. C. to 22.degree. C. The fermentation was harvested
after 70 hours of induction.
[0493] To determine the relative yields of single and 3-copy
clones, clone 201-55-001-VU, containing one genomic integrant of
the single copy pPROT55-V vector, was fermented in parallel with
clone 201-55M3-003-VU, containing one genomic integrant of the
3-copy pPROT55M3-V vector. The fermentations were carried out as
described above except that the pH of the culture was maintained at
5.7 throughout induction. The harvested supernatants from
201-55-001-VU and 201-55M3-003-VU fermentations were analyzed by
gradient SDS-PAGE followed by Colloidal Blue staining (FIG. 8),
which showed that higher proprotein expression was obtained from
the multicopy 201-55M3-003-VU clone compared to the single copy
201-55-001-VU clone. SDS-PAGE results were confirmed with HIC-HPLC
analysis of proprotein concentration in the fermentation
supernatants, demonstrating that the 201-55M3-003-VU clone produced
approximately 600 mg/L of the secreted proprotein while the
201-55-001-VU clone produced approximately 400 mg/L. Thus, the
multicopy 201-55M3-003-VU clone containing three expression
cassettes produced approximately 50% more proprotein compared to
single copy 201-55-001-VU clone containing a single expression
cassette.
[0494] Two conversion strategies were tested using the
201-55M3-003-VU supernatant produced from the fermentation. These
strategies generally follow the strategies described for other
proprotein variants in Example 5, except that they were performed
on a larger scale. In the first strategy, the proprotein was
captured from the supernatant by cation exchange chromatography,
followed by conversion to the mature protein and polish
chromatography. In the second strategy, the proprotein was
converted to the mature protein prior to purification, followed by
capture using cation exchange chromatography, extended conversion
to remove the N-terminal variants, and further polish
chromatography. Both strategies also included an extended
incubation step in a buffer at pH 8.0 after conversion to effect
the selective degradation of N-terminal variants.
[0495] Using the first strategy, capture of the proprotein followed
by conversion and PRT-201 polish purification were effected as
follows. The 201-55M3-003-VU supernatant was harvested from the
fermentation culture and frozen at -80.degree. C. Approximately 7 L
of frozen clarified supernatant (3.5 L from the 201-55M3-003-VU
fermentation described above and 3.5 L from 201-55M3-003-VU shaker
flask cultures prepared generally as described in Examples 4 and 5)
was thawed and diluted 8-fold with deionized water and 1 M sodium
citrate, pH 4.3, at 2-8.degree. C. to obtain a final concentration
of 25 mM sodium citrate. The pH of the solution was adjusted to
4.7. The solution was loaded onto a 2.3 L bed Macroprep High S
cation exchange column at 76 cm/hr at 2-8.degree. C. The column was
washed with 5 CVs of 25 mM sodium citrate, pH 4.7, followed by 5
CVs of 160 mM sodium chloride, 25 mM sodium citrate, pH 4.7. The
proprotein was eluted with 15 CVs of a linear gradient starting
from 160 mM sodium chloride to 500 mM sodium chloride in 25 mM
sodium citrate, pH 4.7, at 87 ml/min (67 cm/hr). The eluate was
collected in fractions. Fractions were analyzed by SDS-PAGE for
protein content (FIG. 9). A small amount of spontaneously converted
protein was observed by SDS-PAGE. Fractions containing the
proprotein were pooled for further processing. The pooled material
was subjected to HIC-HPLC analysis, which showed that it consisted
of 92% proprotein and 8% mature PRT-201.
[0496] To initiate conversion, the pooled material was buffer
exchanged using tangential flow filtration into 100 mM sodium
chloride, 20 mM Tris, pH 4.0, using constant volume diafiltration
at 10-12.degree. C. Tangential flow filtration was performed with
regenerated cellulose membranes, a transmembrane pressure of 15
psi, a crossflow rate of 20 L/min and a flux of 800 mL/min. Three
diavolumes of the buffer at 2-8.degree. C. was added at the same
rate as the flux. Subsequently, three additional volumes of buffer
at ambient temperature were added at the same rate as the flux to
raise the temperature of the conversion solution to the target of
26.degree. C. Tangential flow filtration was used to concentrate
the conversion solution to the target of 1.5 mg/mL using the
conditions of 15 psi transmembrane pressure, 1.2 L/min crossflow
rate, and 76 ml/min flux. However, after approximately 2 minutes of
starting the concentration procedure, unexpected precipitation was
observed and the concentration process was halted. The protein
concentration of the conversion solution was determined to be 1.1
mg/mL by UV absorbance at 280 nm in a volume of 570 mL. To minimize
further precipitation, the conversion solution was diluted to 1
mg/mL with 100 mM sodium chloride, 20 mM Tris, pH 4.0, and filtered
through a 0.22 micron membrane. Sixteen mL of 3 M Tris, pH 9.0 was
added to the conversion solution. The conversion solution was
placed in a water bath at 26.degree. C. The conversion reaction was
monitored by HIC-HPLC analysis. After 30 minutes, HIC-HPLC showed
that the majority of the proprotein had been converted to PRT-201
and some N-terminal variants (FIG. 10). After 1 hour, the
conversion reaction consisted of 0% proprotein, 86% full-length
PRT-201 and 14% N-terminal variants. The conversion material was
incubated further for 4 more hours, at which time HIC-HPLC analysis
showed that the conversion material consisted of 98% full-length
PRT-201 and 2% N-terminal variants.
[0497] The conversion material was diluted 4-fold with deionized
water and 1 M sodium citrate, pH 4.3, to a final concentration of
25 mM sodium citrate. The pH of the solution was adjusted to 5.0 in
preparation for loading onto the polish column. The solution was
loaded onto a 600 mL bed Macroprep High S cation exchange column at
27 mL/min (83 cm/hr). The column was washed with 5 CVs of 20 mM
sodium citrate, pH 5.0 followed by 5 CVs of 160 mM sodium chloride,
20 mM sodium citrate, pH 5.0. PRT-201 was eluted with 15 CVs of a
linear gradient starting from 160 mM to 500 mM sodium chloride in
25 mM sodium citrate, pH 5.0, at 87 mL/min (67 cm/hr). The eluate
was collected in fractions. Fractions were analyzed by SDS-PAGE for
protein content and by SLAP assay for specific activity. Fractions
containing PRT-201 with a specific activity of .gtoreq.30 U/mg were
pooled. The pooled PRT-201 fractions were diafiltered by tangential
flow filtration into formulation buffer (0.1.times.PBS, pH 5.0).
The pH of diafiltered PRT-201 was adjusted to pH 7.4 and the
protein concentration was adjusted to 1 mg/mL by tangential flow
filtration. Vials were filled and lyophilized as described for
PRT-201 produced from the 201-24-266-VU clone in Example 4.
[0498] Using the second strategy, proprotein conversion followed by
purification of PRT-201 was effected as follows. Approximately 7 L
of the frozen clarified supernatant from the 201-55M3-003-VU
fermentation described above was thawed and the conversion reaction
was initiated by tangential flow filtration with a conversion
buffer containing 100 mM Tris, pH 8.0, using constant volume
diafiltration at ambient temperature with regenerated cellulose
membranes. Two diavolumes of 100 mM Tris-HCl, pH 8.0 were
sufficient to change the pH of the retentate from pH 5.0 to 8.0 and
effect conversion. An experiment was also performed by directly
adjusting the pH of the clarified supernatant to 8.0 by adding a
Tris base to a final concentration of 100 mM and adjusting the pH
to 8.0 with 1 N sodium hydroxide. This resulted in the formation of
precipitates and a cloudy supernatant, possibly due to
precipitation of broth components, which was undesirable. The
preferred method of conversion using tangential flow filtration did
not result in precipitation.
[0499] The conversion reaction was monitored by real-time HIC-HPLC
analysis. After initiation of conversion, pro-PRT-201 converted to
mature PRT-201 slowly over the first two hours, followed by an
acceleration of conversion (FIG. 11). At 4.5 hours, the conversion
reaction consisted of approximately 4% pro-PRT-201, 75% mature
PRT-201 and 21% N-terminal variants. At 6.5 hours, the conversion
reaction consisted of approximately 1% pro-PRT-201, 83% mature
PRT-201 and 16% N-terminal variants. The reduction in N-terminal
variants from 21% to 16% from 4.5 hrs to 6.5 hrs may be due to
degradation of N-terminal variants by full length, active PRT-201,
but this was not complete and not as rapid as the reduction in
N-terminal variants seen during conversion of purified pro-PRT 201.
In this second strategy, extending the conversion reaction may not
entirely remove the N-terminal variants due to the competing
proteins in the supernatant that compete for the active site of
elastase. To improve the conversion reaction, the postconversion
material was captured and subsequently diafiltered into an
appropriate buffer to initiate extended conversion at pH 8.0 for
selective degradation of N-terminal variants as described
below.
[0500] Capture of PRT-201 from the conversion material was effected
as follows. The conversion material was buffer exchanged into 20 mM
sodium citrate, pH 5.0 and loaded onto a Macro-Prep High S
cation-exchange chromatography column. The column was washed with 5
CVs of 20 mM sodium citrate, pH 5.0 followed by 5 CVs of 20 mM
sodium citrate, 160 mM sodium chloride, pH 5.0. PRT-201 was eluted
with a linear gradient from 160 mM to 500 mM sodium chloride in 20
mM sodium citrate, pH 5.0. The fractions were analyzed by SDS-PAGE
for protein content, HIC-HPLC for N-terminal variants, UV
absorbance at 280 nm for protein concentration, and SLAP assay for
elastase activity. Two predominant proteins bands were detected by
SDS-PAGE, corresponding to PRT-201 and PRT-201 glycoforms, as shown
in FIG. 12. Fractions that contained PRT-201 glycoforms as
determined by SDS-PAGE or N-terminal variants as determined by
HIC-HPLC were excluded from pooling. Fractions that exhibited
relatively low specific activity (less than 30 U/mg) as determined
by SLAP assay were also excluded from pooling. The remaining
fractions containing PRT-201 were pooled for further processing.
HIC-HPLC analysis of the pooled fractions revealed that this
material consisted of approximately 98% full-length PRT-201 and
1-2% N-terminal variants. The pooled material was stored at
2-8.degree. C. for 12-16 hrs.
[0501] Given the prior observation that N-terminal variants
appeared to decrease over a prolonged conversion period as
described above, the pooled PRT-201 material was subjected to an
extended incubation step at pH 8.0. The extended incubation was
performed by diafiltration and tangential flow filtration with 100
mM Tris, 300 mM sodium chloride, pH 8.0 for 2.5 hours at ambient
temperature. After 2.5 hours, the conversion material consisted of
100% mature PRT-201 as shown by HIC-HPLC analysis. The conversion
material was subjected to diafiltration and tangential flow
filtration into 20 mM sodium citrate, pH 5.0 to suppress elastase
activity and prepare for column chromatography. The diafiltered
PRT-201 material was stored for approximately 64 hours at
2-8.degree. C.
[0502] Polish chromatographic purification of PRT-201 was effected
as follows. The diafiltered PRT-201 material was loaded onto a
Macro-Prep High S cation exchange column and washed with 5 CVs of
Buffer C (20 mM sodium citrate, pH 5.0) followed by 5 CVs of 160 mM
sodium chloride, 20 mM sodium citrate, pH 5.0. Elution of PRT-201
was performed with a linear gradient of 15 CVs starting from 68%
Buffer C and 32% Buffer D (160 mM sodium chloride, 25 mM sodium
citrate, pH 5.0) to 0% Buffer C and 100% Buffer D (500 mM sodium
chloride, 25 mM sodium citrate, pH 5.0), at 50 ml/min (153 cm/hr).
The eluate was collected in fractions. PRT-201 eluted as a
symmetrical peak at 37 mS/cm (330 mM sodium chloride). Fractions
were analyzed by SDS-PAGE analysis for protein content, UV
absorbance at 280 nm for protein concentration and SLAP assay for
specific activity. Fractions containing PRT-201 that had a specific
activity of 30.1-38.8 U/mg were pooled. The pooled PRT-201
fractions were diafiltered by tangential flow filtration into
formulation buffer (0.1.times.PBS, pH 5.0). The pH of diafiltered
PRT-201 was adjusted to pH 7.4 and the protein concentration was
adjusted to 1 mg/mL by tangential flow filtration. Vials were
filled and lyophilized as described for PRT-201 produced from the
201-24-266-VU clone in Example 4.
[0503] The conditions for the conversion procedures described above
were chosen based on conversion optimization studies that examined
protein concentration, temperature, buffer composition, diavolume
and pH variables. In the first study, the effect of proprotein
concentration on the production of N-terminal variants during
conversion was analyzed. Purified pro-PRT-201 from the
201-55M3-003-VU clone (pro-PRT-201-55M3-003-VU) at a starting
concentration of 0.2 mg/mL in 20 mM sodium phosphate, pH 5.0 was
aliquotted and concentrated to 1.0, 1.6, and 1.8 mg/mL using
centrifugal concentrating devices as determined by UV absorbance at
280 nm. Conversion of the proprotein was effected by adding Tris
and sodium chloride to 100 mM each of the four concentration
samples, adjusting the pH from 5.0 to 8.0, and incubating the
samples at ambient temperature. The conversion reaction was
monitored by HIC-HPLC in real-time until the proprotein was
.ltoreq.1% of the total protein (FIG. 13). At this endpoint, the
0.2 mg/mL sample consisted of approximately 8% N-terminal variants,
whereas the 1.0 mg/mL, 1.6 and 2.0 mg/mL samples consisted of
approximately 14%, 19% and 29% N-terminal variants, respectively.
The remainder of the protein in each sample consisted of
full-length PRT-201. These results demonstrate that increasing
concentrations of pro-PRT-201-55-003-VU during conversion leads to
the formation of more N-terminal variants and less full-length
PRT-201. Other studies have suggested that pro-PRT-201-55M3-003-VU
conversion occurs through both intramolecular and intermolecular
reactions. Thus, for this variant proprotein, it is likely that
intramolecular reactions, which are favored in more dilute
proprotein solutions, give rise to more accurate conversion whereas
intermolecular reactions, favored in more concentrated proprotein
solutions, result in less accurate conversion, i.e., the formation
of a higher percentage of the N-terminal variants relative to
full-length PRT-201.
[0504] In the second study, the effect of temperature on the
production of N-terminal variants during conversion was analyzed.
Purified pro-PRT-201 from the 201-55M3-VU clone (named
pro-PRT-201-55M3-003-VU) was produced at a concentration of 1.6
mg/mL was subjected to conversion as described above at either
15.degree. C. or 26.degree. C. The conversion reactions were
monitored in real-time by HIC-HPLC and allowed to progress until
the proprotein comprised <1% of total protein. The time required
to reach this reduction in proprotein was approximately 30 minutes
at 26.degree. C. and approximately 90 minutes at 15.degree. C. At
these times, a similar percentage of N-terminal variants (about 20%
of total protein) for both temperatures was observed. Thus, the
higher temperature of 26.degree. C. resulted in a more rapid
conversion reaction compared to the lower temperature of 15.degree.
C. while producing an essentially identical reaction product
profile.
[0505] The third study examined the effect of buffer composition on
proprotein solubility during the conversion reaction. Purified
pro-PRT-201 (pro-PRT-201-55M3-003-VU) at a concentration of 1.0
mg/mL was subjected to conversion under the conditions listed in
Table 6. The conversion reactions were performed at ambient
temperature except for one (buffer composition of 20 mM Tris-HCl,
100 mM sodium chloride, pH 4.0) that was performed at 2 to
8.degree. C. Conversion reactions were inspected visually for
precipitation. As noted in Table 6, buffer compositions that did
not result in precipitation included 100 mM Tris-HCl, pH 8.0; 100
mM Tris-HCl, 100 mM sodium chloride, pH 5.0; and 100 mM Tris-HCl,
300 mM sodium chloride, pH 8.0. Buffer compositions with lower
concentrations of Tris or without sodium chloride at lower pH
(i.e., pH 5.0) exhibited precipitation.
TABLE-US-00006 TABLE 6 Effect of buffer composition on
precipitation during conversion. Soluble (No Precipitation
Precipitation Buffer Composition Temperature Observed Observed) 1
mM Tris-HCl, pH 5.0 Ambient + 25 mM Tris-HCl, pH 5.0 Ambient + 100
mM Tris-HCl, pH 5.0 Ambient + 100 mM Tris-HCl, pH 8.0 Ambient + 20
mM Tris-HCl, 100 mM sodium chloride, pH 4.0 2-8.degree. C. + 100 mM
Tris-HCl, 100 mM sodium chloride, pH 5.0 Ambient + 100 mM Tris-HCl,
300 mM sodium chloride, pH 8.0 Ambient +
[0506] In the fourth study, the effect of tangential flow
filtration diavolume number on precipitation during conversion of
supernatant containing proprotein (pro-PRT-201-55M3-003-VU) was
analyzed. The solution used for buffer exchange was 100 mM
Tris-HCl, 100 mM sodium chloride, pH 8.0. Additionally, a direct pH
adjustment of the supernatant from pH 5.0 to 8.0 without tangential
flow filtration was tested. As noted in Table 7, direct pH
adjustment of the supernatant resulted in a large amount of
precipitation. With 1 diavolume of exchange, some precipitation was
observed. No precipitation was observed when 2 to 5 diavolumes were
used.
TABLE-US-00007 TABLE 7 Effect of diavolume number on precipitation
during buffer exchange. Diavolumes Precipitation Observed 0 Major
precipitation 1 Minor precipitation 2 No precipitation 3 No
precipitation 5 No precipitation
[0507] In the fifth study, the effect of pH on elastase activity of
mature PRT-201 was analyzed. This study was designed to identify a
useful pH range for conversion that would not result in an
irreversible loss of elastase activity of the mature PRT-201
conversion product. Solutions of 1 mg/mL PRT-201 in 20 mM Tris-HCl,
20 mM potassium phosphate were prepared from pH 1 to 14. Solutions
were kept at ambient temperature for 0.5, 2, 24 and 48 hours. At
the indicated time points, the solutions were tested for elastase
activity in the SLAP assay. Elastase activity was largely stable
from pH 3 to 8 at all time points. At pHs less than 3 and greater
8, elastase activity was reduced at all time points, with a
correlation between longer time points and lower elastase activity.
These results indicated that a conversion reaction performed
outside a pH range of 3 to 8 could negatively impact elastase
activity of the PRT-201 conversion product.
6.8 Example 7
Production of Auto-Activated Recombinant Porcine Type I Pancreatic
Elastase
[0508] A vector encoding auto-activated porcine ELA-1 proenzyme was
expressed P. pastoris. The resulting auto-activated porcine ELA-1
was compared to a porcine ELA-1 protein expressed as a
trypsin-activated wild-type proprotein.
[0509] To construct the trypsin-activated porcine ELA-1 vector, the
porcine ELA-1 coding region was synthesized by Blue Heron
Biotechnology (Bothell, Wash.) using a non-PCR "long oligo"
technique under license from Amgen (Thousand Oaks, Calif.). The
recombinant gene was cloned into the Blue Heron pUC vector, a
derivative of pUC119. The porcine ELA-1 gene was sequenced on both
strands to confirm the correct sequence. SacI and XbaI restriction
sites were incorporated as potential cloning sites flanking the
porcine ELA-1 gene as shown in FIG. 14. A second stop codon was
also added immediately after the native stop codon to minimize
potential ribosome read through. FIG. 15 shows the nature identical
amino acid sequence of porcine ELA-1 proenzyme, which contains the
trypsin-activated site.
[0510] The coding region of porcine ELA-1 was amplified by PCR
using a pair of oligonucleotides containing XhoI and SacI
restriction sites to facilitate cloning. The PCR product was
digested with XhoI and SacI and purified by agarose gel
electrophoresis. The porcine ELA-1 fragment was cloned into the
PV-1 vector at XhoI and SacI restriction sites. The E. coli TOP 10
strain was transformed with the ligation mixture. The cell mixture
was plated on low salt LB plates supplemented with 25 mg/mL Zeocin.
Drug-resistant clones were picked and plasmid DNA was prepared
(Qiagen, Calif.). Based on restriction analysis, a clone containing
the porcine ELA-1 gene insert was identified and the vector was
named pPROT101-24-V. The coding sequence of this vector was
confirmed by DNA sequencing. The cloning scheme for pPROT101-24-V
is depicted in FIG. 16.
[0511] Using the trypsin-activated pPROT101-24-V vector, three
different auto-activated clones were engineered by changing the
trypsin cleavage site in the pro-peptide region of porcine ELA-1 to
elastase cleavage sites. Site-directed mutagenesis was performed
generally as described in Example 4 using synthetic oligonucleotide
primers containing the desired mutations as described in Table 8.
All the mutations in the pro-peptide region were confirmed by
double-stranded DNA sequencing.
TABLE-US-00008 TABLE 8 Cleavage domain sequences of
trypsin-activated and auto-activated porcine ELA-1 vectors.
##STR00001## Mutagenized codons are shaded. The cleaved bond is
between P1 and P'1.
[0512] The wild-type NRRL Y-11430 P. pastoris strain was
transformed and drug-resistant transformants were cultured and
screened for expression of the porcine ELA-1 proproteins as
described in Example 3. Based on analysis by SDS-PAGE and Coomassie
staining (see FIG. 17 and FIG. 18), the wild-type trypsin-activated
pPROT101-24-V clones had the highest levels of expression compared
to the auto-activated clones. Of the auto-activated clones, the
pPROT101-49-V clones had the highest level of expression, followed
by the pPROT101-55L-V clones and then the pPROT101-49-V clones.
Auto-activated pPROT101-42-V and pPROT101-55L-V proproteins
exhibited substantial spontaneous conversion during induction,
while the trypsin-activated pPROT101-24-V and auto-activated
pPROT101-49-V proproteins showed greater stability in the induction
media.
[0513] Studies of the elastase activity of PRT-102 produced by
trypsin activation of proelastase protein expressed from
pPROT101-24-V showed higher specific activity than PRT-201 as shown
in Table 9 below:
TABLE-US-00009 TABLE 9 Elastase activity of three different samples
of mature porcine type I pancreatic elastase (trypsin activated) as
compared to mature human type I pancreatic elastase. Sample Name
PRT-201 PRT-102 PRT-102 PRT-102 Activity as measured 34.6 91.8 99.4
100.7 by SLAP 32.9 88.3 91.3 88.6 (U/mg protein) (Replicates)
Average of Replicates 33.6 88.5 93.5 92.9 Standard Deviation 0.9
3.2 5.2 6.7
[0514] A small-scale conversion experiment was used to determine if
pPROT101-55L-V and pPROT101-49-V proproteins could be converted to
mature enzymes exhibiting elastase activity. pPROT101-55L-V and
pPROT101-49-V shaker flask supernatants were concentrated 10-fold
with an centrifugal filter unit and diluted 5-fold with 100 mM
Tris-HCl, pH 9.0. The conversion was allowed to proceed at room
temperature and elastase activity was monitored over time by SLAP
assay. The average change in absorbance per minute was determined
from each time point and reported as non-normalized reaction
velocity (FIG. 19). Conversion of both pPROT101-49-V and
pPROT101-55L-V supernatants resulted in an increase in elastase
activity. The final time point samples from each clone were
analyzed by SDS-PAGE followed by Coomassie staining and compared to
pre-conversion samples (FIG. 20). The SDS-PAGE results confirmed
that nearly all of the pPROT101-49-V proprotein was converted to
mature protein by the end of the conversion assay. The SDS-PAGE
results also showed that nearly all of the pPROT101-55L-V had been
spontaneously converted to mature protein prior to the conversion
assay.
[0515] Purified preparations of pPROT101-24-V and pPROT101-49-V
proproteins and mature enzymes were submitted to Danforth Plant
Science Center, MO for intact molecular weight analysis. The
proproteins were purified by cation exchange chromatography
(Macroprep High S, Bio-Rad). For mature enzyme analysis, the
proproteins from both clones were first treated to produce mature
enzymes and then purified by cation exchange chromatography. The
trypsin-activated proprotein was treated with trypsin while the
auto-activated proprotein was converted in the presence of 100 mM
Tris-HCl, pH 9.0, followed by cation exchange chromatography. The
major peaks obtained from mass spectrometry analysis are listed in
Table 10.
TABLE-US-00010 TABLE 10 Expected and observed molecular weights for
porcine ELA-1 proteins. Protein Expected MW Observed MW
Trypsin-activated pPROT101-24-V 27068 27064 proprotein
Trypsin-activated pPROT101-24-V 25908 25898 mature enzyme
Auto-activated pPROT101-49-V 27049 27047 proprotein Auto-activated
pPROT101-49-V 25908 25910 mature enzyme
[0516] SDS-PAGE, elastase activity and mass spectrometry results
demonstrated that auto-activated forms of type I porcine pancreatic
elastase can be produced by engineering the pro-peptide sequence to
replace the trypsin cleavage site with an elastase cleavage site.
The expression levels of these auto-activated forms of type I
porcine pancreatic elastase are lower than the wild-type
trypsin-activated form. Of the autoactivated clones tested, those
with the pPROT101-49-V pro-peptide sequence showed the highest
level of expression and the least spontaneous conversion.
Conversion of the pPROT101-49-V and pPROT101-55L-V clones resulted
in the production of mature proteins with substantial elastase
activity. Mass spectrometry analysis revealed that the molecular
weights of pPROT101-49-V proprotein and mature porcine type I
corresponded to the expected masses.
6.9 Example 8
Trypsin Activity Analysis of Mature Recombinant Human Elastase-1 by
Benz Colorimetric Peptide Substrate Assay
[0517] A calorimetric hydrolysis assay using the small peptide
substrate N-benzoyl-Phe-Val-Arg-pNitroanilide (BENZ) was performed
to determine if purified mature elastase protein produced by the
auto-activated clone 201-55M3-003-VU possesses trypsin activity.
Three vials of lyophilized PRT-201 purified from clone
201-55M3-003-VU were retrieved from -80.degree. C. storage and
reconstituted with water to obtain 1 mg/mL PRT-201 in
0.1.times.PBS, pH 7.4. Protein concentrations were confirmed by
measuring UV absorbance at 280 nm. A TrypZean stock solution (10
mg/mL) was used to generate a standard curve for trypsin activity.
A previously tested trypsin-activated PRT-201 sample was included
as a positive control. In addition, some experimental and control
samples were spiked with TrypZean to determine trypsin activity
recovery in the presence of PRT-201. A subset of the spiked and
unspiked samples was treated with soybean trypsin inhibitor (SBTI)
to determine the ability of SBTI to inhibit any intrinsic or spiked
trypsin activity. TrypZean standards were also treated with SBTI to
confirm the effectiveness of the inhibitor. See Table 11 below for
a summary of the samples included in this study.
TABLE-US-00011 TABLE 11 TrypZean dilutions for the standard curve
were prepared using the assay buffer (0.1M Tris, pH 8.3). The
standard curve for TrypZean solutions is shown in FIG. 21. Plus
TrypZean spike Plus SBTI Plus TrypZean Description No addition (to
100 ng/mL) (to 10 ug/mL) spike and SBTI PRT-201 from clone 55M3
Vial #1 PRT-201 from clone 55M3 Vial #2 PRT-201 from clone 55M3
Vial #3 PRT-201 from trypsin activated clone Buffer only (0.1M
Tris, pH 8.3) Not done Not done TrypZean standard, 1.56 ng/mL Not
done Not done TrypZean standard, 3.13 ng/mL Not done Not done
TrypZean standard, 6.25 ng/mL Not done Not done TrypZean standard,
12.5 ng/mL Not done Not done Tr5ypZean standard, 25 ng/mL Not done
Not done TrypZean standard, 50 ng/mL Not done Not done TrypZean
standard, 100 ng/mL Not done Not done
[0518] The substrate solution was prepared (0.4 mg/mL
N-benzoyl-Phe-Val-Arg-pNitroanilide Lot 7733 in 0.1 M Tris, pH 8.3)
and prewarmed to 30.degree. C. in a water bath. In triplicate, 100
microliters of each sample was pipetted into a 96-well microplate.
Using a multichannel pipettor, 200 microliters of substrate
solution was pipetted into each well and the microplate was
immediately placed into a microplate reader preheated to 30.degree.
C. The microplate reader recorded the absorbance at 405 nm for each
well once per minute for 60 minutes.
[0519] The PRT-201 samples were also tested for elastase activity
in the SLAP assay. The SLAP substrate solution was prepared (4.5
mg/mL SLAP in 0.1 M Tris, pH 8.3) and prewarmed to 30.degree. C. in
a water bath. The 1 mg/mL PRT-201 samples were diluted 20.times.
with water to 0.05 mg/mL. In triplicate, 10 microliters of each
sample dilution was pipetted into a 96-well microplate. Using a
multichannel pipettor, 300 microliters of SLAP substrate solution
was pipetted into each well and the microplate was immediately
placed into a microplate reader preheated to 30.degree. C. The
microplate reader recorded the absorbance at 405 nm for each well
once per minute for 5 minutes.
[0520] The results of the BENZ and SLAP activity assays are
respectively presented in Tables 11 and 12 below.
TABLE-US-00012 TABLE 12 Mean trypsin activity, reported as TrypZean
concentration equivalent (ng/mL). [a] Coefficient of regression
<0.8. Plus TrypZean spike Plus SBTI Plus TrypZean Description No
addition (to 100 ng/mL) (to 10 ug/mL) spike and SBTI PRT-201 from
clone 55M3 Vial #1 <1.56 [a] 118.7 <1.56 [a] <1.56 [a]
PRT-201 from clone 55M3 Vial #2 <1.56 [a] 122.4 <1.56 [a]
<1.56 [a] PRT-201 from clone 55M3 Vial #3 <1.56 [a] 122.8
<1.56 [a] <1.56 [a] PRT-201 from trypsin activated clone 8.7
130.0 <1.56 [a] <1.56 [a] Buffer only (0.1M Tris, pH 8.3) 1.3
Not done <1.56 [a] Not done TrypZean standard, 1.56 ng/mL 2.7
Not done <1.56 [a] Not done TrypZean standard, 3.13 ng/mL 3.7
Not done <1.56 [a] Not done TrypZean standard, 6.25 ng/mL 6.5
Not done <1.56 [a] Not done TrypZean standard, 12.5 ng/mL 11.2
Not done <1.56 [a] Not done Tr5ypZean standard, 25 ng/mL 23.5
Not done <1.56 [a] Not done TrypZean standard, 50 ng/mL 49.4 Not
done <1.56 [a] Not done TrypZean standard, 100 ng/mL 102.4 Not
done <1.56 [a] Not done
TABLE-US-00013 TABLE 13 Mean SLAP activity, reported as U/mg
Description No addition PRT-201 from clone 55M3 Vial #1 34.9
PRT-201 from clone 55M3 Vial #2 36.6 PRT-201 from clone 55M3 Vial
#3 35.7 PRT-201 from trypsin activated clone 32.1
[0521] The level of trypsin activity of PRT-201 from clone
201-55M3-003-VU was below the range of the standard curve in the
trypsin activity assay (<1.56 ng/mL). Additionally, the
coefficients of regression for the triplicate hydrolysis reactions
were poor (<0.8), further supporting the absence of trypsin
activity in this auto-activated mature elastase protein. In
contrast, the level of trypsin activity of the control
trypsin-activate sample was determined to be 8.7 ng/mL.
6.10 Example 9
Drug Product Formulation
[0522] A study was conducted to identify lyophilized PRT-201
formulations that had the following characteristics: strong ionic
strength buffer upon reconstitution; pH stability at 7.4; capable
of one-step reconstitution; improved stability; and improved cake
appearance. A screen conducted to assess various pre-formulation
buffers and excipients. Formulations with sodium phosphate buffer
and trehalose and mannitol as excipients were found to meet the
study objectives. One specific lyophilized formulation having these
characteristics produces upon reconstitution with water a 4 mg/mL
pH 7.4 solution of PRT-201 in 25 mM sodium phosphate, 3% mannitol,
and 1% trehalose.
[0523] Thus, in certain embodiments, a formulation of the
disclosure contains sodium phosphate buffer and/or trehalose and/or
mannitol. Such formulations can be modified, e.g., by the addition
of sodium chloride, so as to produce a more isotonic solution upon
reconstitution. Alternatively, the formulation can be modified
following reconstitution so as to produce a more isotonic solution.
In specific embodiments, the formulation is a unit dosage. In other
embodiments, the formulation has a molarity ranging from 120 to 150
mmol/L, more preferably ranging from 135 to 140 mmol/L (for example
138 mmol/L).
TABLE-US-00014 7. SEQUENCE LISTING SEQ ID NO. Description Type of
sequence Sequence 1 Mature human Amino acid, single
VVGGTEAGRNSWPSQISLQYRSGGSRYHTCGGTL elastase I, letter format,
wherein: IRQNWVMTAAHCVDYQKTFRVVAGDHNLSQNDGT including first X = V
or L EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV "valine"
TLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN
GQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTM
VCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGV
TSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN 2 Mature human Amino acid,
single VGGTEAGRNSWPSQISLQYRSGGSRYHTCGGTLI elastase I, letter
format, wherein: RQNWVMTAAHCVDYQKTFRVVAGDHNLSQNDGT minus first X =
V or L EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV "valine"
TLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN
GQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTM
VCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGV
TSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN 3 Mature human Amino acid,
single GGTEAGRNSWPSQISLQYRSGGSRYHTCGGTLIR elastase I, letter
format, wherein: QNWVMTAAHCVDYQKTFRVVAGDHNLSQNDGTE minus first two
X = V or L QYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSVT "valines"
LNSYVQLGVLPQEGAILANNSPCYITGWGKTKTNG
QLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTMV
CAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVT
SFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN 4 Mature human Amino acid,
single AVGGTEAGRNSWPSQISLQYRSGGSRYHTCGGTL elastase I, letter
format, wherein: IRQNWVMTAAHCVDYQKTFRVVAGDHNLSQNDGT with first X =
V or L EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV "valine"
TLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN substituted by
GQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTM "alanine"
VCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGV
TSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN 5 Mature human Amino acid,
single VVGGTEAGRNSWPSQISLQYRSGGSRYHTCGGTL elastase I letter format
IRQNWVMTAAHCVDYQKTFRVVAGDHNLSQNDGT (isotype 2),
EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV including first
TLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN "valine"
GQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTM VCAGGDGSSLWMPG 6 Engineered
Amino acid, single TQDLPETNAAVVGGTEAGRNSWPSQISLQYRSGG elastase
letter format, wherein SRYHTCGGTLIRQNVVVMTAAHCVDYQKTFRVVAG
proprotein no. X = V or L DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 1
(pPROT42 DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI variant)
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV
NGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN 7 Engineered Amino
acid, single TQDLPETNAAAVGGTEAGRNSWPSQISLQYRSGG elastase letter
format, wherein: SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG proprotein no.
X = V or L DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 2
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV
NGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN 8 Engineered Amino
acid, single TQDLPETAAAVVGGTEAGRNSWPSQISLQYRSGG elastase letter
format, wherein: SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG proprotein no.
X = V or L DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 3
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV
NGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN 9 Engineered Amino
acid, single TQDLPETNNAPVGGTEAGRNSWPSQISLQYRSGG elastase letter
format, wherein: SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG proprotein no.
X = V or L DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 4
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV
NGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN 10 Wild-type Amino
acid, single TQDLPETNARVVGGTEAGRNSWPSQISLQYRSGG elastase letter
format, wherein: SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG proprotein no.
X = V or L DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 5 (produced
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI from
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY pPROT24
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV trypsin
NGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS activated WINNVIASN sequence)
11 Consensus Amino acid, three Xaa.sub.1 Xaa.sub.2 Xaa.sub.3
elastase letter format Xaa.sub.1 = alanine, leucine, isoleucine,
recognition methionine, lysine, asparagine or valine sequence 1
Xaa.sub.2 = proline, alanine, leucine, isoleucine, (Positions
glycine, valine, or threonine Xaa.sub.1 = P3, Xaa.sub.3 = alanine,
leucine, valine, isoleucine, Xaa.sub.2 = P2, or serine Xaa.sub.3 =
P1) 12 Consensus Amino acid, three Xaa.sub.1 Pro Xaa.sub.2 elastase
letter format Xaa.sub.1 = alanine, leucine, isoleucine, recognition
methionine, lysine, or valine sequence 2 Xaa.sub.2 = alanine,
leucine, valine, isoleucine, (Positions P3- or serine P2-P1) 13
Consensus Amino acid, three Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 elastase
letter format Xaa.sub.1 = asparagine or alanine recognition
Xaa.sub.2 = proline or alanine sequence 3 Xaa.sub.3 = alanine or
leucine or valine (Positions P3- P2-P1) 14 Elastase Amino acid,
three Ala Ala Ala recognition letter format sequence 1 (Positions
P3- P2-P1) 15 Elastase Amino acid, three Asn Ala Ala recognition
letter format sequence 2 (Positions P3- P2-P1) 16 Elastase Amino
acid, three Asn Ala Pro recognition letter format sequence 3
(Positions P3- P2-P1) 17 Wild-type Amino acid, three Asn Ala Arg
trypsin letter format recognition sequence (pPROT24) (Positions P3-
P2-P1) 18 Elastase Amino acid, three Ala Pro Ala recognition letter
format sequence 5 (Positions P3- P2-P1) 19 Elastase Amino acid,
three Ala Ala Pro recognition letter format sequence 6 (Positions
P3- P2-P1) 20 Elastase Amino acid, three Asn Pro Ala recognition
letter format sequence 7 (Positions P3- P2-P1 of Variants 48 and
55) 21 Elastase Amino acid, three Leu Pro Ala recognition letter
format sequence 8 22 Human Amino acid, three Thr Gln Asp Leu Pro
Glu Thr Asn Ala Arg elastase letter format activation sequence 1
(Wild-type) 23 Human Amino acid, three Thr Gln Asp Leu Pro Glu Thr
Asn Ala Ala elastase letter format activation sequence 2 24
pro-PROT- Amino acid, three Thr Asn Ala Arg Val Val Gly Gly 201
cleavage letter format site 25 pPROT40 Amino acid, three Thr Ala
Ala Ala Val Val Gly Gly cleavage site letter format 26 pPROT41
Amino acid, three Thr Asn Ala Ala Ala Val Gly Gly cleavage site
letter format 27 pPROT42 Amino acid, three Thr Asn Ala Ala Val Val
Gly Gly cleavage site letter format 28 pPROT43 Amino acid, three
Thr Asn Ala Pro Val Val Gly Gly cleavage site letter format 29
pPROT44 Amino acid, three Thr Gly Ala Gly Ile Val Gly Gly cleavage
site letter format 30 pPROT45 Amino acid, three Thr Val Pro Gly Val
Val Gly Gly cleavage site letter format 31 pPROT46 Amino acid,
three Thr Ala Pro Gly Val Val Gly Gly cleavage site letter format
32 pPROT47 Amino acid, three Thr Asn Pro Gly Val Val Gly Gly
cleavage site letter format 33 Coding region Nucleotide
ACCCAGGACCTTCCGGAAACCAATGCCCGCGTA of a human
GTCGGAGGGACTGAGGCCGGGAGGAATTCCTG elastase-1
GCCCTCTCAGATTTCCCTCCAGTACCGGTCTGG (NCBI
AGGTTCCCGGTATCACACCTGTGGAGGGACCCT Accession No.
TATCAGACAGAACTGGGTGATGACAGCTGCTCA NM_001971)
CTGCGTGGATTACCAGAAGACTTTCCGCGTGGT GGCTGGAGACCATAACCTGAGCCAGAATGATGG
CACTGAGCAGTACGTGAGTGTGCAGAAGATCGT GGTGCATCCATACTGGAACAGCGATAACGTGGC
TGCCGGCTATGACATCGCCCTGCTGCGCCTGGC
CCAGAGCGTTACCCTCAATAGCTATGTCCAGCTG
GGTGTTCTGCCCCAGGAGGGAGCCATCCTGGCT AACAACAGTCCCTGCTACATCACAGGCTGGGGC
AAGACCAAGACCAATGGGCAGCTGGCCCAGACC CTGCAGCAGGCTTACCTGCCCTCTGTGGACTAC
GCCATCTGCTCCAGCTCCTCCTACTGGGGCTCC ACTGTGAAGAACACCATGGTGTGTGCTGGTGGA
GATGGAGTTCGCTCTGGATGCCAGGGTGACTCT GGGGGCCCCCTCCATTGCTTGGTGAATGGCAAG
TATTCTGTCCATGGAGTGACCAGCTTTGTGTCCA
GCCGGGGCTGTAATGTCTCCAGGAAGCCTACAG
TCTTCACCCAGGTCTCTGCTTACATCTCCTGGAT AAATAATGTCATCGCCTCCAACTGA 34
Yeast alpha Amino acid, three
Met-Arg-Phe-Pro-Ser-Ile-Phe-Thr-Ala-Val-Leu-Phe- factor signal
letter format Ala-Ala-Ser-Ser-Ala-Leu-Ala-Ala-Pro-Val-Asn-Thr-
peptide 35 20F primer Nucleotide
ggctcgagaaaagagaggctgaagctactcaggaccttccggaaac caatgcccgg 36 24R
primer Nucleotide gggccgcggcttatcagttggaggcgatgacat 37 pPROT42 P3
Amino acid, single AAVVGGTEAGRNSWPSQISLQYRSGGSRYHTCG cleavage site
letter format, wherein: GTLIRQNWVMTAAHCVDYQKTFRVVAGDHNLSQN variant
X = V or L DGTEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLA elastase
QSVTLNSYVQLGVLPQEGAILANNSPCYITGWGKT
KTNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVK
NTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSX
HGVTSFVSSRGCNVSRKPTVFTQVSAYISWINNVIA SN 38 pPROT42 P2 Amino acid,
single AVVGGTEAGRNSWPSQISLQYRSGGSRYHTCGGT cleavage site letter
format, wherein: LIRQNWVMTAAHCVDYQKTFRVVAGDHNLSQNDG variant X = V
or L TEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQS
elastase VTLNSYVQLGVLPQEGAILANNSPCYITGWGKTKT
NGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNT
MVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHG
VTSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN 39 Mature Amino acid, single
VVGGTEAQRNSWPSQISLQYRSGSSWAHTCGGTL porcine letter format
IRQNWVMTAAHCVDRELTFRVVVGEHNLNQNDGT pancreatic
EQYVGVQKIVVHPYWNTDDVAAGYDIALLRLAQSV elastase I
TLNSYVQLGVLPRAGTILANNSPCYITGWGLTRTN (from
GQLAQTLQQAYLPTVDYAICSSSSYWGSTVKNSM GenBank
VCAGGDGVRSGCQGDSGGPLHCLVNGQYAVHGV Accession
TSFVSRLGCNVTRKPTVFTRVSAYISWINNVIASN P00772.1) 40 Elastase Amino
acid, three Glu Thr Ala Ala Ala Val Val Gly variant letter format
propeptide cleavage domain 40 41 Elastase Amino acid, three Glu Thr
Asn Ala Ala Ala Val Gly variant letter format propeptide cleavage
domain 41 42 Elastase Amino acid, three Glu Thr Asn Ala Ala Val Val
Gly variant letter format propeptide cleavage domain 42 43 Elastase
Amino acid, three Glu Thr Asn Ala Pro Val Val Gly variant letter
format propeptide cleavage domain 43 44 Elastase Amino acid, three
Glu Thr Gly Ala Gly Ile Val Gly variant letter format propeptide
cleavage domain 44 45 Elastase Amino acid, three Glu Thr Val Pro
Gly Val Val Gly variant letter format propeptide cleavage domain 45
46 Elastase Amino acid, three Glu Thr Ala Pro Gly Val Val Gly
variant letter format propeptide cleavage domain 46 47 Elastase
Amino acid, three Glu Thr Asn Pro Gly Val Val Gly variant letter
format propeptide cleavage domain 47 48 Elastase Amino acid, three
Glu Thr Asn Pro Ala Val Val Gly variant letter format propeptide
cleavage domain 48 49 Elastase Amino acid, three Glu Thr Asn His
Ala Val Val Gly variant letter format propeptide cleavage domain 49
50 Yeast alpha- Amino acid, three
Met-Arg-Phe-Pro-Ser-Ile-Phe-Thr-Ala-Val-Leu-Phe- mating factor
letter format Ala-Ala-Ser-Ser-Ala-Leu-Ala-Ala-Pro-Val-Asn-Thr-
signal peptide, Thr-Thr-Glu-Asp-Glu-Thr-Ala-Gln-Ile-Pro-Ala-Glu-
propeptide, Ala-Val-Ile-Gly-Tyr-Leu-Asp-Leu-Glu-Gly-Asp-Phe- and
spacer Asp-Val-Ala-Val-Leu-Pro-Phe-Ser-Asn-Ser-Thr-Asn- sequence 1
Asn-Asn-Gly-Leu-Leu-Phe-Ile-Asn-Thr-Thr-Ile-Ala-
Ser-Ile-Ala-Ala-Lys-Glu-Glu-Gly-Val-Ser-Leu-Asp- 51 Yeast alpha-
Amino acid, three Met-Arg-Phe-Pro-Ser-Ile-Phe-Thr-Ala-Val-Leu-Phe-
mating factor letter format
Ala-Ala-Ser-Ser-Ala-Leu-Ala-Ala-Pro-Val-Asn-Thr- signal peptide
Thr-Thr-Glu-Asp-Glu-Thr-Ala-Gln-Ile-Pro-Ala-Glu- and
Ala-Val-Ile-Gly-Tyr-Leu-Asp-Leu-Glu-Gly-Asp-Phe- propeptide
Asp-Val-Ala-Val-Leu-Pro-Phe-Ser-Asn-Ser-Thr-Asn- sequence 2
Asn-Asn-Gly-Leu-Leu-Phe-Ile-Asn-Thr-Thr-Ile-Ala-
Ser-Ile-Ala-Ala-Lys-Glu-Glu-Gly-Val-Ser-Leu-Asp- 52 Elastase Amino
acid, three Glu Thr Lys Pro Ala Val Val Gly variant letter format
propeptide cleavage domain 52 53 Elastase Amino acid, three Glu Thr
His Pro Ala Val Val Gly variant letter format propeptide cleavage
domain 53 54 Elastase Amino acid, three Glu His Asn Pro Ala Val Val
Gly variant letter format propeptide cleavage domain 54 55 Elastase
Amino acid, three His Thr Asn Pro Ala Val Val Gly variant letter
format propeptide cleavage domain 55 56 Elastase Amino acid, three
Pro Thr His Pro Ala Val Val Gly variant letter format propeptide
cleavage domain 56 57 Elastase Amino acid, three Pro Thr Asn Pro
Ala Val Val Gly variant letter format propeptide cleavage domain 57
58 Elastase Amino acid, three His Thr His Pro Ala Val Val Gly
variant letter format propeptide cleavage domain 58 59 Elastase
Amino acid, three Glu Thr Phe Pro Ala Val Val Gly variant letter
format propeptide cleavage domain 59 60 Elastase Amino acid, three
His Thr Phe Pro Ala Val Val Gly variant letter format propeptide
cleavage domain 60 61 Elastase Amino acid, three Gly Thr Phe Pro
Ala Val Val Gly variant letter format propeptide cleavage domain 61
62 Elastase Amino acid, three His Thr Gly Pro Ala Val Val Gly
variant letter format propeptide cleavage domain 62 63 Elastase
Amino acid, three His Thr Lys Pro Ala Val Val Gly variant letter
format propeptide cleavage domain 63 64 Elastase Amino acid, single
TQDLPETNPAVVGGTEAGRNSWPSQISLQYRSGG proenzyme letter format,
wherein: SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG with variant X = V or L
DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 48
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV
NGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN 65 Elastase Amino
acid, single TQDLPETNHAVVGGTEAGRNSWPSQISLQYRSGG proenzyme letter
format, wherein: SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG with variant X
= V or L DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 49
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV
NGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS 66 Elastase Amino acid, single
TQDLPETKPAVVGGTEAGRNSWPSQISLQYRSGG proenzyme letter format,
wherein: SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG with variant X = V or L
DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 52
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV
NGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN 67 Elastase Amino
acid, single TQDLPETHPAVVGGTEAGRNSWPSQISLQYRSGG proenzyme letter
format, wherein: SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG with variant X
= V or L DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 53
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV
NGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN 68 Elastase Amino
acid, single TQDLPEHNPAVVGGTEAGRNSWPSQISLQYRSGG proenzyme letter
format, wherein: SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG with variant X
= V or L DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 54
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV
NGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN 69 Elastase Amino
acid, single TQDLPHTNPAVVGGTEAGRNSWPSQISLQYRSGG proenzyme letter
format, wherein: SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG with variant X
= V or L DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 55
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV
NGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN 70 Wild-type Amino
acid, single ARVVGGTEAGRNSWPSQISLQYRSGGSRYHTCG elastase letter
format, wherein: GTLIRQNWVMTAAHCVDYQKTFRVVAGDHNLSQN +AlaArg X = V
or L DGTEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLA cleavage
QSVTLNSYVQLGVLPQEGAILANNSPCYITGWGKT variant
KTNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVK
NTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSX
HGVTSFVSSRGCNVSRKPTVFTQVSAYISWINNVIA SN 71 Wild-type Amino acid,
single RVVGGTEAGRNSWPSQISLQYRSGGSRYHTCGG elastase +Arg letter
format, wherein: TLIRQNWVMTAAHCVDYQKTFRVVAGDHNLSQND cleavage X = V
or L GTEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQ variant
SVTLNSYVQLGVLPQEGAILANNSPCYITGWGKTK
TNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNT
MVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHG
VTSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN 72 Variant 48 Amino acid,
three Thr Gln Asp Leu Pro Glu Thr Asn Pro Ala human letter format
elastase activation peptide 73 Variant 55 Amino acid, three Thr Gln
Asp Leu Pro His Thr Asn Pro Ala human letter format elastase
activation peptide 74 Human Amino acid, three Xaa.sub.1 Xaa.sub.2
Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6 Xaa.sub.7 Xaa.sub.8
elastase letter format Xaa.sub.1 = glutamate, histidine,
proline,
glycine, cleavage asparagine, lysine, or alanine domain Xaa.sub.2 =
threonine, alanine, proline or histidine consensus Xaa.sub.3 =
alanine, leucine, isoleucine, methionine, sequence; lysine,
asparagine or valine corresponds Xaa.sub.4 = proline, alanine,
leucine, isoleucine, glycine, to residues valine, or threonine P5,
P4, P3, Xaa.sub.5 = alanine, leucine, valine, isoleucine, or serine
P2, P1, P'1, Xaa.sub.6 = alanine, leucine, valine, isoleucine or
serine P'2, and P'3 of Xaa.sub.7 = glycine, alanine, or valine an
elastase Xaa.sub.8 = valine, threonine, phenylalanine, tyrosine, or
cleavage tryptophan domain 75 PCR Nucleic Acid ATC TAC GTA GTC GGA
GGG ACT GAG GCC mutagenesis primer 76 PCR Nucleic Acid gtc gac aag
ctt atc agt tgg agg cga t mutagenesis primer 77 Mature ELA1
Protein, single letter VVGGTEAGRNSWPSQISLQYRSGGSRYHTCGGTL
C-terminal format IRQNWVMTAAHCVDYQKTFRVVAGDHNLSQNDGT variant of
EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV Talas et al.
TLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN
GQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTM VCAGGDGVRSGCQGDSGGPPPLLGEWQVFSPW
SDQLCVQPGL 78 Mature ELA-1 Protein, single letter
VVGGTEAGRNSWPSQISLQYRSGGSBYHTCGGTL variants format, wherein:
IRQNWVJTAAHCVDYQKTFRVVAGDHNLSQNDGT B = W or R
EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV J = M or V
TLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN X = V or L
GQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTM Z = Q or R
VCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGV
TSFVSSRGCNVSRKPTVFTZVSAYISWINNVIASN 79 Activation Protein, single
letter TUDLPETNAR peptide format, wherein variants (wild- U = Q or
H type, trypsin cleavable) 80 Activation Protein, three letter Thr
Xaa.sub.1 Asp Leu Pro Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5
Xaa.sub.6 peptide format Xaa.sub.1 = glutamine or histidine variant
Xaa.sub.2 = glutamate, histidine, proline, glycine, consensus
asparagine, lysine, or alanine Xaa.sub.3 = threonine, alanine,
proline or histidine Xaa.sub.4 = alanine, leucine, isoleucine,
methionine, lysine, asparagine or valine Xaa.sub.5 = proline,
alanine, leucine, isoleucine, glycine, valine, or threonine
Xaa.sub.6 = alanine, leucine, valine, isoleucine, or serine 81
Coding region Nucleotide ACTCAGGACCTTCCGGAAACCAATGCCCGGGTA of
ELA-1.2A GTCGGAGGGACTGAGGCCGGGAGGAACTCCTG
GCCCTCTCAGATTTCCCTCCAGTACCGGTCTGG AGGTTCCTGGTATCACACCTGTGGAGGGACCCT
TATCAGACAGAACTGGGTGATGACAGCTGCACA CTGCGTGGATTACCAGAAGACTTTCCGCGTGGT
GGCTGGAGACCATAACCTGAGCCAGAATGATGG CACTGAGCAGTACGTGAGTGTGCAGAAGATCGT
GGTGCATCCATACTGGAACAGCGATAACGTGGC TGCAGGCTATGACATCGCCCTGCTGCGCCTGGC
CCAGAGCGTTACCCTCAATAGCTATGTCCAGCTG
GGTGTTCTGCCCCAGGAGGGAGCCATCCTGGCT AACAACAGTCCCTGCTACATCACAGGCTGGGGC
AAGACCAAGACCAATGGGCAGCTGGCCCAGACC TTGCAGCAGGCTTACCTGCCCTCTGTGGACTAT
GCCATCTGCTCCAGCTCCTCCTACTGGGGCTCC ACTGTGAAGAACACTATGGTGTGTGCTGGTGGA
GATGGAGTTCGCTCTGGATGTCAGGGTGACTCT GGGGGCCCCCTCCATTGCTTGGTGAATGGCAAG
TATTCTCTTCATGGAGTGACCAGCTTTGTGTCCA
GCCGGGGCTGTAATGTCTCTAGAAAGCCTACAG
TCTTCACACGGGTCTCTGCTTACATCTCCTGGAT AAATAATGTCATCGCCTCCAACTGATAA 82
Translation Protein, single letter
TQDLPETNARVVGGTEAGRNSWPSQISLQYRSGG product of format
SWYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG ELA-1.2A
DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY (trypsin
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI activated
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY pPROT24
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV sequence)
NGKYSLHGVTSFVSSRGCNVSRKPTVFTRVSAYIS WINNVIASN 83 Variants of
Protein, single letter translation format, wherein: product of U =
Q or H TUDLPETNARVVGGTEAGRNSWPSQISLQYRSGG ELA-1.2A B = W or R
SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG (trypsin J = M or V
DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY activated X = V or L
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI pPROT24 Z = Q or R
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY sequence)
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV
NGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS WINNVIASN 84 Mature human Amino
acid, single VVGGTEAGRNSWPSQISLQYRSGGSBYHTCGGTL elastase I, letter
format, wherein: IRQNWVJTAAHCVDYQKTFRVVAGDHNLSQNDGT including first
B = W or R EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV "valine" J = M or V
TLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN X = V or L
GQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTM Z = Q or R
VCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGV
TSFVSSRGCNVSRKPTVFTZVSAYISWINNVIASN 85 Mature human Amino acid,
single VGGTEAGRNSWPSQISLQYRSGGSBYHTCGGTLI elastase I, letter
format, wherein: RQNWVJTAAHCVDYQKTFRVVAGDHNLSQNDGTE minus first B =
W or R QYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSVT "valine" J = M or V
LNSYVQLGVLPQEGAILANNSPCYITGWGKTKTNG X = V or L
QLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTMV Z = Q or R
CAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVT
SFVSSRGCNVSRKPTVFTZVSAYISWINNVIASN 86 Mature human Amino acid,
single GGTEAGRNSWPSQISLQYRSGGSBYHTCGGTLIR elastase I, letter
format, wherein: QNWVJTAAHCVDYQKTFRVVAGDHNLSQNDGTE minus first two
B = W or R QYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSVT "valines" J = M or V
LNSYVQLGVLPQEGAILANNSPCYITGWGKTKTNG X = V or L
QLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTMV Z = Q or R
CAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVT
SFVSSRGCNVSRKPTVFTZVSAYISWINNVIASN 87 Mature human Amino acid,
single AVGGTEAGRNSWPSQISLQYRSGGSBYHTCGGTL elastase I, letter
format, wherein: IRQNWVJTAAHCVDYQKTFRVVAGDHNLSQNDGT with first B =
W or R EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV "valine" J = M or V
TLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN substituted by X = V or L
GQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTM "alanine" Z = Q or R
VCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGV
TSFVSSRGCNVSRKPTVFTZVSAYISWINNVIASN 88 Engineered Amino acid,
single TUDLPETNAAVVGGTEAGRNSWPSQISLQYRSGG elastase letter format,
wherein: SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG proprotein no. U = Q or
H DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 1 (pPROT42 B = W or R
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI variant) J = M or V
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY X = V or L
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV Z = Q or R
NGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS WINNVIASN 89 Engineered Amino
acid, single TUDLPETNAAAVGGTEAGRNSWPSQISLQYRSGG elastase letter
format, wherein: SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG proprotein no.
U = Q or H DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 2 B = W or R
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI J = M or V
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY X = V or L
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV Z = Q or R
NGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS WINNVIASN 90 Engineered Amino
acid, single TUDLPETAAAVVGGTEAGRNSWPSQISLQYRSGG elastase letter
format, wherein: SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG proprotein no.
U = Q or H DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 3 B = W or R
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI J = M or V
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY X = V or L
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV Z = Q or R
NGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS WINNVIASN 91 Engineered Amino
acid, single TUDLPETNNAPVGGTEAGRNSWPSQISLQYRSGG elastase letter
format, wherein: SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG proprotein no.
U = Q or H DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 4 B = W or R
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI J = M or V
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY X = V or L
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV Z = Q or R
NGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS WINNVIASN 92 Engineered Amino
acid, single TUDLPETNARVVGGTEAGRNSWPSQISLQYRSGG elastase letter
format, wherein: SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG proprotein no.
U = Q or H DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 5 (pPROT24 B = W or R
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI trypsin J = M or V
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY activated X = V or L
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV sequence) Z = Q or R
NGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS WINNVIASN 93 Consensus Amino
acid, three Xaa.sub.1 Pro Xaa.sub.2 elastase letter format
Xaa.sub.1 = alanine, leucine, isoleucine, methionine, recognition
lysine, asparagine or valine sequence 2 Xaa.sub.2 = alanine,
leucine, valine, isoleucine, or serine (Positions P3- P2-P1) 94
pPROT42 P3 Amino acid, single AAVVGGTEAGRNSWPSQISLQYRSGGSBYHTCG
cleavage site letter format, wherein:
GTLIRQNWVJTAAHCVDYQKTFRVVAGDHNLSQN variant B = W or R
DGTEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLA elastase J = M or V
QSVTLNSYVQLGVLPQEGAILANNSPCYITGWGKT X = V or L
KTNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVK Z = Q or R
NTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSX
HGVTSFVSSRGCNVSRKPTVFTZVSAYISWINNVIA SN 95 pPROT42 P2 Amino acid,
single AVVGGTEAGRNSWPSQISLQYRSGGSBYHTCGGT cleavage site letter
format, wherein: LIRQNWVJTAAHCVDYQKTFRVVAGDHNLSQNDG variant B = W
or R TEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQS elastase J = M or V
VTLNSYVQLGVLPQEGAILANNSPCYITGWGKTKT X = V or L
NGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNT Z = Q or R
MVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHG
VTSFVSSRGCNVSRKPTVFTZVSAYISWINNVIASN 96 Yeast alpha- Amino acid,
three Met-Arg-Phe-Pro-Ser-Ile-Phe-Thr-Ala-Val-Leu-Phe- mating
factor letter format
Ala-Ala-Ser-Ser-Ala-Leu-Ala-Ala-Pro-Val-Asn-Thr- signal peptide,
Thr-Thr-Glu-Asp-Glu-Thr-Ala-Gln-Ile-Pro-Ala-Glu- propeptide,
Ala-Val-Ile-Gly-Tyr-Ser-Asp-Leu-Glu-Gly-Asp-Phe- and spacer
Asp-Val-Ala-Val-Leu-Pro-Phe-Ser-Asn-Ser-Thr-Asn- sequence
Asn-Gly-Leu-Leu-Phe-Ile-Asn-Thr-Thr-Ile-Ala-Ser-Ile-
Ala-Ala-Lys-Glu-Glu-Gly-Val-Ser-Leu-Glu-Lys-Arg- Glu-Ala-Glu-Ala 97
Yeast alpha- Amino acid, three
Met-Arg-Phe-Pro-Ser-Ile-Phe-Thr-Ala-Val-Leu-Phe- mating factor
letter format Ala-Ala-Ser-Ser-Ala-Leu-Ala-Ala-Pro-Val-Asn-Thr-
signal peptide Thr-Thr-Glu-Asp-Glu-Thr-Ala-Gln-Ile-Pro-Ala-Glu- and
Ala-Val-Ile-Gly-Tyr-Ser-Asp-Leu-Glu-Gly-Asp-Phe- propeptide
Asp-Val-Ala-Val-Leu-Pro-Phe-Ser-Asn-Ser-Thr-Asn- sequence
Asn-Gly-Leu-Leu-Phe-Ile-Asn-Thr-Thr-Ile-Ala-Ser-Ile-
Ala-Ala-Lys-Glu-Glu-Gly-Val-Ser-Leu-Glu- 98 Elastase Amino acid,
single TUDLPETNPAVVGGTEAGRNSWPSQISLQYRSGG proenzyme letter format,
wherein: SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG with variant U = Q or H
DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage B = W or R
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 48 J = M or V
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY X = V or L
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV Z = Q or R
NGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS WINNVIASN 99 Elastase Amino
acid, single TUDLPETNHAVVGGTEAGRNSWPSQISLQYRSGG proenzyme letter
format, wherein: SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG with variant U
= Q or H DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage B = W or R
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 49 J = M or V
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY X = V or L
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV Z = Q or R
NGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS WINNVIASN 100 Elastase Amino
acid, single TUDLPETKPAVVGGTEAGRNSWPSQISLQYRSGG proenzyme letter
format, wherein: SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG with variant U
= Q or H DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage B = W or R
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 52 J = M or V
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY X = V or L
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV Z = Q or R
NGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS WINNVIASN 101 Elastase Amino
acid, single TUDLPETHPAVVGGTEAGRNSWPSQISLQYRSGG
proenzyme letter format, wherein:
SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG with variant U = Q or H
DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage B = W or R
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 53 J = M or V
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY X = V or L
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV Z = Q or R
NGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS WINNVIASN 102 Elastase Amino
acid, single TUDLPEHNPAVVGGTEAGRNSWPSQISLQYRSGG proenzyme letter
format, wherein: SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG with variant U
= Q or H DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage B = W or R
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 54 J = M or V
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY X = V or L
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV Z = Q or R
NGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS WINNVIASN 103 Elastase Amino
acid, single TUDLPHTNPAVVGGTEAGRNSWPSQISLQYRSGG proenzyme letter
format, wherein: SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG with variant U
= Q or H DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage B = W or R
DIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 55 J = M or V
TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY X = V or L
WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV Z = Q or R
NGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS WINNVIASN 104 Wild-type Amino
acid, single ARVVGGTEAGRNSWPSQISLQYRSGGSBYHTCG elastase letter
format, wherein: GTLIRQNWVJTAAHCVDYQKTFRVVAGDHNLSQN +AlaArg B = W
or R DGTEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLA cleavage J = M or V
QSVTLNSYVQLGVLPQEGAILANNSPCYITGWGKT variant X = V or L
KTNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVK Z = Q or R
NTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSX
HGVTSFVSSRGCNVSRKPTVFTZVSAYISWINNVIA SN 105 Wild-type Amino acid,
single RVVGGTEAGRNSWPSQISLQYRSGGSBYHTCGGT elastase +Arg letter
format, wherein: LIRQNWVJTAAHCVDYQKTFRVVAGDHNLSQNDG cleavage B = W
or R TEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQS variant J = M or V
VTLNSYVQLGVLPQEGAILANNSPCYITGWGKTKT X = V or L
NGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNT Z = Q or R
MVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHG
VTSFVSSRGCNVSRKPTVFTZVSAYISWINNVIASN 106 Mature human Amino acid,
single TEAGRNSWPSQISLQYRSGGSBYHTCGGTLIRQN elastase I, letter
format, wherein: WVJTAAHCVDYQKTFRVVAGDHNLSQNDGTEQYV minus N B = W
or R SVQKIVVHPYWNSDNVAAGYDIALLRLAQSVTLNS terminal J = M or V
YVQLGVLPQEGAILANNSPCYITGWGKTKTNGQLA "VVGG" X = V or L
QTLQQAYLPSVDYAICSSSSYWGSTVKNTMVCAG sequence Z = Q or R
GDGVRSGCQGDSGGPLHCLVNGKYSXHGVTSFV SSRGCNVSRKPTVFTZVSAYISWINNVIASN
107 Mature human Amino acid, single
AGRNSWPSQISLQYRSGGSBYHTCGGTLIRQNWV elastase I, letter format,
wherein: JTAAHCVDYQKTFRVVAGDHNLSQNDGTEQYVSV minus N B = W or R
QKIVVHPYWNSDNVAAGYDIALLRLAQSVTLNSYV terminal J = M or V
QLGVLPQEGAILANNSPCYITGWGKTKTNGQLAQT "VVGGTE" X = V or L
LQQAYLPSVDYAICSSSSYWGSTVKNTMVCAGGD sequence Z = Q or R
GVRSGCQGDSGGPLHCLVNGKYSXHGVTSFVSS RGCNVSRKPTVFTZVSAYISWINNVIASN 108
Mature human Amino acid, single NSWPSQISLQYRSGGSBYHTCGGTLIRQNWVJTA
elastase I, letter format, wherein:
AHCVDYQKTFRVVAGDHNLSQNDGTEQYVSVQKI minus N B = W or R
VVHPYWNSDNVAAGYDIALLRLAQSVTLNSYVQLG terminal J = M or V
VLPQEGAILANNSPCYITGWGKTKTNGQLAQTLQQ "VVGGTEAG X = V or L
AYLPSVDYAICSSSSYWGSTVKNTMVCAGGDGVR R" sequence Z = Q or R
SGCQGDSGGPLHCLVNGKYSXHGVTSFVSSRGC NVSRKPTVFTZVSAYISWINNVIASN 109
FIG. 1A Nucleic Acid GAATTCAGTACTCAGGACCTTCCGGAAACCAATG sequence
CCCGGGTAGTCGGAGGGACTGAGGCCGGGAGG AACTCCTGGCCCTCTCAGATTTCCCTCCAGTACC
GGTCTGGAGGTTCCTGGTATCACACCTGTGGAG GGACCCTTATCAGACAGAACTGGGTGATGACAG
CTGCACACTGCGTGGATTACCAGAAGACTTTCC GCGTGGTGGCTGGAGACCATAACCTGAGCCAGA
ATGATGGCACTGAGCAGTACGTGAGTGTGCAGA AGATCGTGGTGCATCCATACTGGAACAGCGATA
ACGTGGCTGCAGGCTATGACATCGCCCTGCTGC GCCTGGCCCAGAGCGTTACCCTCAATAGCTATG
TCCAGCTGGGTGTTCTGCCCCAGGAGGGAGCCA
TCCTGGCTAACAACAGTCCCTGCTACATCACAGG CTGGGGCAAGACCAAGACCAATGGGCAGCTGG
CCCAGACCTTGCAGCAGGCTTACCTGCCCTCTG
TGGACTATGCCATCTGCTCCAGCTCCTCCTACTG
GGGCTCCACTGTGAAGAACACTATGGTGTGTGC TGGTGGAGATGGAGTTCGCTCTGGATGTCAGGG
TGACTCTGGGGGCCCCCTCCATTGCTTGGTGAA
TGGCAAGTATTCTCTTCATGGAGTGACCAGCTTT
GTGTCCAGCCGGGGCTGTAATGTCTCTAGAAAG
CCTACAGTCTTCACACGGGTCTCTGCTTACATCT
CCTGGATAAATAATGTCATCGCCTCCAACTGATA AGCTTGGATCCGTCGAC 110 FIG. 1A
Amino Acid, single MKRILAIHQAMEGAPRVTLTSRANSISTSTHHSVLH sequence
letter format SGAPVGGAGADGIVHRGQVSLLQGLGQLPIGLGLA
PACDVAGTVVSQDGSLLGQNTQLDIAIEGNALGQA
QQGDVIACSHVIAVPVWMHHDLLHTHVLLSAIILAQ
VMVSSHHAESLLVIHAVCSCHHPVLSDKGPSTGVI
PGTSRPVLEGNLRGPGVPPGLSPSDYPGIGFRKVL S 111 FIG. 1B Nucleic Acid
ACTATTGCCAGCATTGCTGCTAAAGAAGAAGGG sequence
GTATCTCTCGAGAAAAGAGAGGCTGAAGCTACT CAGGACCTTCCGGAAACCAATGCCCGGGTAGTC
GGGGGG 112 FIG. 1B Amino Acid, three THR ILE ALA SER ILE ALA ALA
LYS GLU GLU GLY sequence letter format VAL SER LEU GLU LYS ARG GLU
ALA GLU ALA THR GLN ASP LEU PRO GLU THR ASN ALA ARG VAL VAL GLY GLY
113 FIG. 13 Nucleic Acid CCGCGGACCCAGGACTTTCCAGAAACCAACGCC sequence
CGGGTAGTTGGAGGGACCGAGGCTCAGAGGAA TTCTTGGCCATCTCAGATTTCCCTCCAGTACCGG
TCTGGAAGTTCGTGGGCTCACACCTGTGGAGGG ACCCTCATCAGGCAGAACTGGGTGATGACAGCC
GCTCACTGCGTGGACAGAGAGTTGACCTTCCGT GTGGTGGTTGGAGAGCACAACCTGAACCAGAAC
GATGGCACCGAGCAGTACGTGGGGGTGCAGAA GATCGTGGTGCATCCCTACTGGAACACCGACGA
CGTGGCTGCAGGCTATGACATCGCCCTGCTGCG CCTGGCCCAGAGTGTAACCCTCAACAGCTACGT
CCAGCTGGGTGTTCTGCCAAGGGCTGGGACCAT CCTGGCTAACAACAGTCCCTGCTACATCACAGG
GTGGGGCCTGACCAGGACCAATGGGCAGCTGG CCCAGACCCTGCAGCAGGCTTACCTGCCCACCG
TGGACTACGCCATCTGCTCCAGCTCCTCGTACT GGGGCTCCACCGTGAAGAACAGCATGGTGTGCG
CCGGAGGGGACGGAGTTCGCTCTGGATGTCAG GGTGATTCTGGGGGCCCCCTTCATTGCTTGGTG
AATGGTCAGTATGCTGTCCACGGTGTAACCAGCT
TCGTGTCCCGCCTGGGCTGTAATGTCACCAGGA
AGCCCACAGTCTTCACCAGGGTCTCTGCTTACAT
CTCTTGGATAAATAACGTCATTGCCAGCAACTGA TAATCTAGA 114 FIG. 14 Amino
Acid, single TQDFPETNARVVGGTEAQRNSWPSQISLQYRSGS sequence letter
format SWAHTCGGTLIRQNWVMTAAHCVDRELTFRVVVG
EHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGY
DIALLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYI
TGWGLTRTNGQLAQTLQQAYLPTVDYAICSSSSY
WGSTVKNSMVCAGGDGVRSGCQGDSGGPLHCLV
NGQYAVHGVTSFVSRLGCNVTRKPTVFTRVSAYIS WINNVIASN 115 Cleavage Amino
Acid, three Phe Pro Glu Thr Asn Ala Arg Val Val Gly domain letter
format sequence of trypsin- activated pPROT101- 24-V 116 Cleavage
Amino Acid, three Phe Pro Glu Thr Asn Ala Ala Val Val Gly domain
letter format sequence of auto-activated pPROT101- 42-V 117
Cleavage Amino Acid, three Leu Pro His Thr Asn Pro Ala Val Val Gly
domain letter format sequence of auto-activated pPROT101- 49-V 118
Cleavage Amino Acid, three Phe Pro Glu Thr Asn His Ala Val Val Gly
domain letter format sequence of auto-activated pPROT101- 55L-V 119
Consensus Amino acid, three Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 elastase
letter format Xaa.sub.1 = any natural amino acid except pro or gly
recognition Xaa.sub.2 = pro, ala, leu, ile, gly, val, his or thr
sequence 5 Xaa.sub.3 = ala, leu, val, ile, or ser (Positions
Xaa.sub.1 = P3, Xaa.sub.2 = P2, Xaa.sub.3 = P1) 120 Consensus Amino
acid, three Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5
sequence 1 letter format Xaa.sub.1 = any natural amino acid for the
Xaa.sub.2 = any natural amino acid except gly, lys, phe, propeptide
tyr, trp, or arg portion of the Xaa.sub.3 = any natural amino acid
except pro or gly cleavage Xaa.sub.4 = pro, ala, leu, ile, gly,
val, his or thr domain Xaa.sub.5 = ala, leu, val, ile, or ser
(Positions Xaa.sub.1 = P5, Xaa.sub.2 = P4, Xaa.sub.3 = P3,
Xaa.sub.4 = P2, Xaa.sub.5 = P1) 121 Consensus Amino acid, three
Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6
Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 sequence 2 letter format Xaa.sub.10
for the Xaa.sub.1 = thr activation Xaa.sub.2 = gln or his peptide
Xaa.sub.3 = asp (Positions Xaa.sub.4 = leu Xaa.sub.1 = P10,
Xaa.sub.5 = pro Xaa.sub.2 = P9, Xaa.sub.6 = any natural amino acid
Xaa.sub.3 = P8, Xaa.sub.7 = any natural amino acid except gly, lys,
phe, Xaa.sub.4 = P7, tyr, trp, or arg Xaa.sub.5 = P6, Xaa.sub.8 =
any natural amino acid except pro or gly Xaa.sub.6 = P5, Xaa.sub.9
= pro, ala, leu, ile, gly, val, his or thr Xaa.sub.7 = P4,
Xaa.sub.10 = ala, leu, val, ile, or ser Xaa.sub.8 = P3, Xaa.sub.9 =
P2, Xaa.sub.10 = P1) 122 Proelastase Amino acid, three Xaa.sub.1
Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6 Xaa.sub.7
Xaa.sub.8 Xaa.sub.9 consensus letter format Xaa.sub.10 Xaa.sub.11
Xaa.sub.12 Xaa.sub.13 sequence 1 Xaa.sub.1 = thr for residues
Xaa.sub.2 = gln or his P10 through Xaa.sub.3 = asp P3' Xaa.sub.4 =
leu Xaa.sub.5 = pro Xaa.sub.6 = any natural amino acid Xaa.sub.7 =
any natural amino acid except gly, lys, phe, tyr, trp, or arg
Xaa.sub.8 = any natural amino acid except pro or gly Xaa.sub.9 =
pro, ala, leu, ile, gly, val, his or thr Xaa.sub.10 = ala, leu,
val, ile, or ser Xaa.sub.11 = ala, leu, val, ile, or ser Xaa.sub.12
= gly, ala, or val Xaa.sub.13 = gly, val, thre, phe, try, or trp
123 Proelastase Amino acid, three Xaa.sub.1 Xaa.sub.2 Xaa.sub.3
Xaa.sub.4 Xaa.sub.5 Xaa.sub.6 Xaa.sub.7 Xaa.sub.8 cleavage letter
format Xaa.sub.1 = glu, his, pro, gly, asn, lys, or ala domain
Xaa.sub.2 = thr, ala, pro or his consensus Xaa.sub.3 = ala, leu,
ile, met, lys, asn or val sequence 2 Xaa.sub.4 = pro, ala, leu,
ile, gly, val, or thr (Positions Xaa.sub.5 = ala, leu, val, ile, or
ser Xaa.sub.1 = P5, Xaa.sub.6 = ala, leu, val, ile, or ser
Xaa.sub.2 = P4, Xaa.sub.7 = gly, ala, or val Xaa.sub.3 = P3,
Xaa.sub.8 = gly, val, thre, phe, try, or trp Xaa.sub.4 = P2,
Xaa.sub.5 = P1, Xaa.sub.6 = P1', Xaa.sub.7 = P2', Xaa.sub.8 =
P3')
124 Consensus Amino acid, three Xaa.sub.1 Xaa.sub.2 Xaa.sub.3
elastase letter format Xaa.sub.1 = ala, leu, ile, met, lys, asn,
his, or val recognition Xaa.sub.2 = pro, ala, leu, ile, gly, val,
or thr sequence 6 Xaa.sub.3 = ala, leu, val, ile, or ser (Positions
P3- P2-P1) 125 Proelastase Amino acid, three Xaa.sub.1 Xaa.sub.2
Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6 Xaa.sub.7 Xaa.sub.8
cleavage letter format Xaa.sub.1 = glu, his, pro, gly, asn, lys, or
ala domain Xaa.sub.2 = thr, ala, pro or his consensus Xaa.sub.3 =
ala, leu, ile, met, lys, asn, thr, or val sequence 3 Xaa.sub.4 =
pro, ala, leu, ile, gly, val, asn, or thr (Positions Xaa.sub.5 =
ala, leu, val, ile, asn, or ser Xaa.sub.1 = P5, Xaa.sub.6 = ala,
leu, val, ile, or ser Xaa.sub.2 = P4, Xaa.sub.7 = gly, ala, or val
Xaa.sub.3 = P3, Xaa.sub.8 = gly, val, thre, phe, try, or trp
Xaa.sub.4 = P2, Xaa.sub.5 = P1, Xaa.sub.6 = P1', Xaa.sub.7 = P2',
Xaa.sub.8 = P3')
8. SPECIFIC EMBODIMENTS, CITATION OF REFERENCES
[0524] The present invention is exemplified by the specific
embodiments below.
[0525] 1. A protein comprising (i) an elastase activation sequence
comprising an elastase recognition sequence operably linked to (ii)
the amino acid sequence of a mature elastase.
[0526] 2. The protein of embodiment 1, wherein the elastase
recognition sequence comprises SEQ ID NO:11.
[0527] 3. The protein of embodiment 1, wherein the elastase
recognition sequence comprises SEQ ID NO:12.
[0528] 4. The protein of embodiment 1, wherein the elastase
recognition sequence comprises SEQ ID NO:13.
[0529] 5. The protein of embodiment 1, wherein the elastase
recognition sequence comprises SEQ ID NO:93.
[0530] 6. The protein of embodiment 1, embodiment 2 or embodiment
4, wherein the elastase recognition sequence comprises any one of
SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID
NO:21.
[0531] 7. The protein of embodiment 1, wherein the activation
sequence comprises SEQ ID NO:80.
[0532] 8. The protein of embodiment 1 or embodiment 7, wherein the
activation sequence comprises SEQ ID NO:23, SEQ ID NO:72, or SEQ ID
NO:73.
[0533] 9. The protein of embodiment 1, wherein the protein
comprises a cleavage domain comprising SEQ ID NO:74.
[0534] 10. The protein of embodiment 1 or embodiment 9, wherein the
cleavage domain comprises any one of SEQ ID NO:42, SEQ ID NO:43,
SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:53, SEQ ID
NO:54 and SEQ ID NO:55.
[0535] 11. The protein of any one of embodiments 1 to 5 and 7 which
comprises SEQ ID NO:64.
[0536] 12. The protein of any one of embodiments 1 to 5 and 7 which
comprises SEQ ID NO:69.
[0537] 13. A type I proelastase protein comprising a cleavage
domain sequence of SEQ ID NO:74.
[0538] 14. The type I proelastase protein of embodiment 13, wherein
the cleavage domain comprises any one of SEQ ID NO:42, SEQ ID
NO:43, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:53, SEQ
ID NO:54 or SEQ ID NO:55.
[0539] 15. The type I proelastase protein of embodiment 13 or
embodiment 14, wherein the mature elastase sequence comprises a
sequence having at least 85% sequence identity to the amino acid
sequence from position 6 (e.g., C-terminal to the P5' residue
according to the elastase amino acid designations herein) to the
end of SEQ ID NO:84 or SEQ ID NO:1.
[0540] 16. The type I proelastase protein of embodiment 13 or
embodiment 14, which comprises a sequence having at least 95%
sequence identity to the amino acid sequence from position 6 (e.g.,
C-terminal to the P5' residue according to the elastase amino acid
designations herein) to the end of SEQ ID NO:84 or SEQ ID NO:1.
[0541] 17. The type I proelastase protein of embodiment 13 or
embodiment 14, which comprises a sequence having at least 98%
sequence identity to the amino acid sequence from position 6 to the
end of SEQ ID NO:84 or SEQ ID NO:1.
[0542] 18. The type I proelastase protein of embodiment 13 or
embodiment 14, wherein the mature elastase comprises a sequence
having up to 10 conservative amino acid changes relative to the
amino acid sequence from position 6 to the end of SEQ ID NO:84 or
SEQ ID NO:1.
[0543] 19. The type I proelastase protein of embodiment 13 or
embodiment 14, which comprises a sequence having up to 7
conservative amino acid changes relative to the amino acid sequence
from position 6 to the end of SEQ ID NO:84 or SEQ ID NO:1.
[0544] 20. The type I proelastase protein of embodiment 13 or
embodiment 14, which comprises a sequence having up to 5
conservative amino acid changes relative to the amino acid sequence
from position 6 to the end of SEQ ID NO:84 or SEQ ID NO:1.
[0545] 21. The type I proelastase protein of any one of embodiments
13 to 20, wherein the amino acid residue denoted by "Xaa.sub.1" in
SEQ ID NO:74 is glutamate or histidine.
[0546] 22. The type I proelastase protein of any one of embodiments
13 to 21, wherein the amino acid residue denoted by "Xaa.sub.4" in
SEQ ID NO:74 is proline.
[0547] 23. The type I proelastase protein of any one of embodiments
13 to 22, wherein the amino acid residue denoted by "Xaa.sub.5" in
SEQ ID NO:74 is alanine.
[0548] 24. The type I proelastase protein of any one of embodiments
13 to 23, wherein the amino acid residue denoted by "Xaa.sub.1" in
SEQ ID NO:74 is histidine, by "Xaa.sub.4" in SEQ ID NO:74 is
proline, and by "Xaa.sub.5" in SEQ ID NO:74 is alanine.
[0549] 25. The type I proelastase protein of any one of embodiments
13 to 24 which comprises the amino acid sequence of SEQ ID
NO:103.
[0550] 26. The type I proelastase protein of embodiment 25 which
comprises the amino acid sequence of SEQ ID NO:64.
[0551] 27. The type I proelastase protein of embodiment 25 which
comprises the amino acid sequence of SEQ ID NO:69.
[0552] 28. The protein of any one of embodiments 1 to 27 which is
isolated.
[0553] 29. The protein of any one of embodiments 1 to 27 which
comprises a signal sequence.
[0554] 30. A protein comprising (i) a signal sequence; (ii) an
elastase activation sequence comprising an elastase recognition
sequence; and (iii) the amino acid sequence of a mature
elastase.
[0555] 31. The protein of embodiment 30, wherein the signal
sequence is operable in Pichia pastoris.
[0556] 32. The protein of embodiment 30, wherein the signal
sequence is a yeast .alpha.-factor signal peptide.
[0557] 33. The protein of embodiment 32, wherein the yeast
.alpha.-factor signal peptide comprises the amino acid sequence of
SEQ ID NO:34.
[0558] 34. The protein of embodiment 30, wherein the signal
sequence is a mammalian secretion signal sequence.
[0559] 35. The protein of embodiment 34, wherein the mammalian
secretion signal sequence is a porcine type I elastase signal
sequence.
[0560] 36. The protein of embodiment 34, wherein the mammalian
secretion signal sequence is a human type I elastase signal
sequence.
[0561] 37. The protein of embodiment 1 or embodiment 30 wherein the
elastase recognition sequence is a type I human elastase
recognition sequence.
[0562] 38. The protein of embodiment 1 or embodiment 30 wherein the
mature elastase is a human type I elastase.
[0563] 39. The protein of embodiment 1 or embodiment 30 wherein the
mature elastase is a porcine type I elastase.
[0564] 40. A nucleic acid encoding a protein of any one of
embodiments 1 to 39.
[0565] 41. A nucleic acid molecule comprising a nucleotide sequence
that encodes a protein, said protein comprising (i) an activation
sequence comprising an elastase recognition sequence operably
linked to (ii) the amino acid sequence of an elastase.
[0566] 42. The nucleic acid molecule of embodiment 41, wherein the
protein further comprises a signal sequence operably linked to said
activation sequence.
[0567] 43. The nucleic acid molecule of embodiment 41 or embodiment
42, wherein the signal sequence is operable in Pichia pastoris.
[0568] 44. The nucleic acid of any one of embodiments 41 to 43,
wherein the elastase recognition sequence is a type I elastase
recognition sequence.
[0569] 45. The nucleic acid of any one of embodiments 41 to 44,
wherein the elastase recognition sequence is a type I human
elastase recognition sequence.
[0570] 46. The nucleic acid molecule of any one of embodiments 41
to 45 wherein the elastase recognition sequence comprises SEQ ID
NO:11.
[0571] 47. The nucleic acid molecule of any one of embodiments 41
to 45 wherein the elastase recognition sequence comprises SEQ ID
NO:12.
[0572] 48. The nucleic acid molecule of any one of embodiments 41
to 45 wherein the elastase recognition sequence comprises SEQ ID
NO:13.
[0573] 49. The nucleic acid molecule of any one of embodiments 41
to 45 wherein the elastase recognition sequence comprises SEQ ID
NO:93.
[0574] 50. The nucleic acid of any one of embodiments 41 to 46 and
48, wherein the elastase recognition sequence comprises any one of
SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID
NO:21.
[0575] 51. The nucleic acid molecule of any one of embodiments 41
to 50, wherein the activation sequence comprises SEQ ID NO:80.
[0576] 52. The nucleic acid molecule of any one of embodiments 41
to 45 and 51, wherein the activation sequence comprises SEQ ID
NO:23, SEQ ID NO:72, or SEQ ID NO:73.
[0577] 53. The nucleic acid molecule of any one of embodiments 41
to 46, wherein the protein comprises a cleavage domain comprising
SEQ ID NO:74.
[0578] 54. The nucleic acid molecule of any one of embodiments 41
to 46 and 53, wherein the cleavage domain comprises any one of SEQ
ID NO:42, SEQ ID NO:43, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:52,
SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55.
[0579] 55. The nucleic acid of any one of embodiments 41 to 54,
wherein the elastase is a mature human type I elastase.
[0580] 56. The nucleic acid of any one of embodiments 41 to 54,
wherein the elastase is a mature porcine type I elastase.
[0581] 57. The nucleic acid of any one of embodiments 41 to 54,
wherein the elastase comprises the amino acid sequence of any one
of SEQ ID NO:5, SEQ ID NO:84, SEQ ID NO:87, and SEQ ID NO:39.
[0582] 58. The nucleic acid of embodiment 57 wherein the elastase
comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID
NO:4.
[0583] 59. The nucleic acid molecule of any one of embodiments 42
to 58, wherein the signal sequence is a yeast .alpha.-factor signal
peptide.
[0584] 60. The nucleic acid molecule of embodiment 59, wherein the
protein comprises the amino acid sequence of any one of SEQ ID
NO:34, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:96 and SEQ ID
NO:97.
[0585] 61. The nucleic acid molecule of any one of embodiments 42
and 44 to 58, wherein the signal sequence is a mammalian secretion
signal sequence.
[0586] 62. The nucleic acid molecule of embodiment 61 wherein the
mammalian secretion signal sequence is a porcine type I elastase
signal sequence.
[0587] 63. The nucleic acid molecule of embodiment 61 wherein the
mammalian secretion signal sequence is a human type I elastase
signal sequence.
[0588] 64. The nucleic acid molecule of any one of embodiments 41
to 63, wherein the protein comprises the amino acid sequence of any
one of SEQ ID NOS.: 88 to 91 and 98 to 103.
[0589] 65. The nucleic acid molecule of embodiment 64, wherein the
protein comprises the amino acid sequence of any one of SEQ ID
NOS.: 6 to 9 and 64 to 69.
[0590] 66. The nucleic acid molecule of embodiment 64, wherein the
protein comprises any of the combinations of elastase polymorphisms
set forth in Table 2.
[0591] 67. The nucleic acid molecule of embodiment 64, wherein the
protein comprises any of the combinations of elastase polymorphisms
set forth in Table 3.
[0592] 68. A protein encoded by the nucleic acid molecule of any
one of embodiments 41 to 67.
[0593] 69. The protein of embodiment 68 which is isolated.
[0594] 70. A vector comprising the nucleic acid molecule of any one
of embodiments 41 to 67.
[0595] 71. The vector of embodiment 70 further comprising a
nucleotide sequence that controls gene expression is operably
linked to the nucleotide sequence which encodes said protein.
[0596] 72. The vector of embodiment 70 or embodiment 71 in which
the nucleotide sequence which encodes said protein is
multimerized.
[0597] 73. A host cell comprising the vector of any one of
embodiments 70 to 72.
[0598] 74. The host cell of embodiment 73 in which at least one
copy of said vector is integrated into the host cell genome.
[0599] 75. The host cell of embodiment 74 in which one copy of said
vector is integrated into the host cell genome.
[0600] 76. The host cell of embodiment 74 in which two to five
copies of said vector are integrated into the host cell genome.
[0601] 77. The host cell of embodiment 74 or 76 in which two copies
of said vector are integrated into the host cell genome.
[0602] 78. The host cell of embodiment 74 or 76 in which three
copies of said vector are integrated into the host cell genome.
[0603] 79. A host cell comprising at least one copy of the nucleic
acid molecule of any one of embodiments 41 to 67 integrated into
its genome.
[0604] 80. The host cell of embodiment 79 in which one copy of said
nucleic acid molecule is integrated into its genome.
[0605] 81. The host cell of embodiment 79 in which two to five
copies of said nucleic acid molecule is integrated into its
genome.
[0606] 82. The host cell of embodiment 79 in which two copies of
said nucleic acid molecule is integrated into its genome.
[0607] 83. The host cell of embodiment 79 in which three copies of
said nucleic acid molecule is integrated into its genome.
[0608] 84. A cell genetically engineered to express the nucleic
acid molecule of any one of embodiments 41 to 67.
[0609] 85. The cell of embodiment 84, wherein the nucleotide
sequence is operably linked to a methanol-inducible promoter.
[0610] 86. A Pichia pastoris cell genetically engineered to express
the nucleotide sequence of any one of embodiments 41 to 67.
[0611] 87. The Pichia pastoris cell of embodiment 87, in which the
nucleotide sequence is operably linked to a methanol inducible
promoter.
[0612] 88. A cell culture supernatant comprising the protein of any
one of embodiments 1 to 27 or embodiment 68.
[0613] 89. A method of producing an elastase protein, comprising
culturing the host cell of embodiment 84 under conditions in which
the protein is produced.
[0614] 90. A method of producing an elastase protein, comprising
culturing the host cell of embodiment 85 under conditions in which
the protein is produced.
[0615] 91. A method of producing an elastase protein, comprising
culturing the host cell of embodiment 86 under conditions in which
the protein is produced.
[0616] 92. A method of producing an elastase protein, comprising
culturing the host cell of embodiment 87 under conditions in which
the protein is produced.
[0617] 93. The method of embodiment 91 or embodiment 92, wherein
said conditions include a period of growth or induction at a pH of
2 to 6.
[0618] 94. The method of embodiment 91 or embodiment 92, wherein
said conditions comprise a period of growth or induction at a
temperature of 22.degree. C. to 28.degree. C.
[0619] 95. The method of any one of embodiments 89 to 92, wherein
the host cell is cultured in complex medium.
[0620] 96. The method of embodiment 95, wherein the complex medium
is buffered methanol-complex medium or buffered glycerol-complex
medium.
[0621] 97. The method of any one of embodiments 89 to 96, wherein
the host cell is cultured in the presence of a citrate, succinate
or acetate compound.
[0622] 98. The method of embodiment 97, wherein the citrate,
succinate or acetate compound is sodium citrate, sodium succinate
or sodium acetate, respectively.
[0623] 99. The method of embodiment 97 or embodiment 98, wherein
one citrate, succinate or acetate compound is present in said
culture at a concentration of 5-50 mM, 7.5-100 mM, 10-150 mM,
50-200 mM, 100-150 mM, 75-125 mM, or 90-110 mM.
[0624] 100. The method of embodiment 97 or embodiment 98, wherein
more than one citrate, succinate or acetate compound is present in
said culture, and wherein the total concentration of citrate,
succinate or acetate compounds in said solution is 5-50 mM, 7.5-100
mM, 10-150 mM, 50-200 mM, 100-150 mM, 75-125 mM, or 90-110 mM.
[0625] 101. The method of any one of embodiments 89 to 100 further
comprising recovering the protein.
[0626] 102. The method of embodiment 95, wherein the protein is
recovered by recovering the supernatant.
[0627] 103. The method of embodiment 95, wherein the protein is
recovered from the supernatant.
[0628] 104. The method of embodiment any one of embodiments 95 to
104, wherein the protein recovered lacks the signal sequence.
[0629] 105. The method of embodiment any one of embodiments 95 to
104, wherein the protein recovered lacks both the signal sequence
and the activation sequence.
[0630] 106. The method of any one of embodiments 89 to 92 and 93 to
105, further comprising raising the pH of a solution containing the
protein to a pH of 6 to 12.
[0631] 107. The method of any one of embodiments 89 to 92 and 93 to
106, which further comprises contacting the protein with a
catalytic amount of an elastase.
[0632] 108. The method of any one of embodiments 89 to 92 and 93 to
106, which further comprises subjecting the protein to
autoactivating conditions, contacting the protein with a catalytic
amount of an elastase, or both.
[0633] 109. The method of embodiment 108, wherein the protein is
subjected to autoactivation conditions.
[0634] 110. The method of embodiment 109, wherein the protein is in
the supernatant when subjected to autoactivation conditions.
[0635] 111. A method of producing an elastase protein, comprising:
[0636] (a) culturing a host cell capable of expressing a
recombinant proelastase protein in the presence of a first citrate,
succinate or acetate compound; [0637] (b) recovering the
recombinant proelastase protein from said host cell culture; and
[0638] (c) optionally, exposing the recombinant proelastase protein
to activation conditions to produce a mature elastase protein.
[0639] thereby producing an elastase protein.
[0640] 112. The method of embodiment 111, wherein the method
comprises the step of exposing the recombinant proelastase protein
to activation conditions to produce a mature elastase protein.
[0641] 113. The method of embodiment 112, wherein the recombinant
proelastase protein is purified prior to said exposure to
activating conditions.
[0642] 114. The method of embodiment 113, wherein said recombinant
proelastase protein is purified in the presence of a second
citrate, succinate or acetate compound, such that a solution
comprising said purified proelastase protein and second citrate,
succinate or acetate compound is produced.
[0643] 115. The method of any one of embodiments 111 to 114,
wherein said first citrate, succinate or acetate compound is sodium
citrate, sodium succinate or sodium acetate, respectively.
[0644] 116. The method of embodiment 114 or embodiment 115, wherein
said second citrate, succinate or acetate compound is sodium
citrate, sodium succinate or sodium acetate, respectively.
[0645] 117. The method of any one of embodiments 114 to 116,
wherein said first and second citrate, succinate or acetate
compound are the same.
[0646] 118. The method of any one of embodiments 114 to 116,
wherein said first and second citrate, succinate or acetate
compound are different.
[0647] 119. The method of any one of embodiments 114 to 118,
wherein one citrate, succinate or acetate compound is present in
said culture at a concentration of 5-50 mM, 7.5-100 mM, 10-150 mM,
50-200 mM, 100-150 mM, 75-125 mM, or 90-110 mM.
[0648] 120. The method of any one of embodiments 114 to 118,
wherein more than one citrate, succinate or acetate compound is
present in said culture, and wherein the total concentration of
citrate, succinate or acetate compounds in said culture is 5-50 mM,
7.5-100 mM, 10-150 mM, 50-200 mM, 100-150 mM, 75-125 mM, or 90-110
mM.
[0649] 121. The method of any one of embodiments 111 to 120,
wherein one citrate, succinate or acetate compound is present in
said solution at a concentration of 5-50 mM, 7.5-100 mM, 10-150 mM,
50-200 mM, 100-150 mM, 75-125 mM, or 90-110 mM.
[0650] 122. The method of any one of embodiments 111 to 120,
wherein more than one citrate, succinate or acetate compound is
present in said solution, and wherein the total concentration of
citrate, succinate or acetate compounds in said solution is 5-50
mM, 7.5-100 mM, 10-150 mM, 50-200 mM, 100-150 mM, 75-125 mM, or
90-110 mM.
[0651] 123. The method of any one of embodiments 111 to 122,
wherein the host cell is cultured in complex medium.
[0652] 124. The method of embodiment 123, wherein the complex
medium is buffered methanol-complex medium or buffered
glycerol-complex medium.
[0653] 125. The method of any one of embodiments 111 to 124 wherein
the proelastase protein is recovered from the supernatant of said
host cell.
[0654] 126. The method of any one of embodiments 111 to 125,
wherein said activation conditions comprise exposure to
trypsin.
[0655] 127. The method of any one of embodiments 111 to 125,
wherein said activation conditions are autoactivation
conditions.
[0656] 128. The method of any one of embodiments 111 to 127 which
further comprises the step of isolating said mature elastase
protein.
[0657] 129. The method of any one of embodiments 111 to 128,
wherein the mature elastase protein is a mature type I elastase
protein.
[0658] 130. The method of embodiment 129, wherein the mature type I
elastase protein is a human type I mature elastase protein.
[0659] 131. The method of embodiment 129, wherein the mature type I
elastase protein is a porcine type I mature elastase protein.
[0660] 132. A method of producing a mature elastase protein,
comprising: [0661] (a) lyophilizing a proelastase protein; [0662]
(b) storing the lyophilized proelastase protein; [0663] (c)
reconstituting the lyophilized proelastase protein; and [0664] (d)
activating the reconstituted proelastase protein, thereby producing
a mature elastase protein.
[0665] 133. The method of embodiment 132, wherein the proelastase
protein is recombinant.
[0666] 134. The method of embodiment 133, wherein the proelastase
protein is made by or obtainable by a process comprising (i)
culturing a host cell that is capable of expressing the proelastase
protein under conditions in which the proelastase protein is
expressed; and (ii) recovering the proelastase protein.
[0667] 135. The method of embodiment 134, wherein the host cell is
cultured in complex medium.
[0668] 136. The method of embodiment 135, wherein the complex
medium is buffered methanol-complex medium or buffered
glycerol-complex medium.
[0669] 137. The method of any one of embodiments 132 to 136,
wherein the host cell is cultured in the presence of a citrate,
succinate or acetate compound.
[0670] 138. The method of embodiment 137, wherein the citrate,
succinate or acetate compound is sodium citrate, sodium succinate
or sodium acetate, respectively.
[0671] 139. The method of embodiment 137 or embodiment 138, wherein
one citrate, succinate or acetate compound is present in said
culture at a concentration of 5-50 mM, 7.5-100 mM, 10-150 mM,
50-200 mM, 100-150 mM, 75-125 mM, or 90-110 mM.
[0672] 140. The method of embodiment 137 or embodiment 138, wherein
more than one citrate, succinate or acetate compound is present in
said culture, and wherein the total concentration of citrate,
succinate or acetate compounds in said solution is 5-50 mM, 7.5-100
mM, 10-150 mM, 50-200 mM, 100-150 mM, 75-125 mM, or 90-110 mM.
[0673] 141. The method of any one of embodiments 132 to 140 wherein
the proelastase protein is recovered from the supernatant of said
host cell.
[0674] 142. The method of any one of embodiments 132 to 141,
wherein the proelastase protein is purified prior to
lyophilization.
[0675] 143. The method of any one of embodiments 132 to 142,
wherein the lyophilized proelastase protein is stored for a period
of at least one day, at least one week, at least one month or at
least three months.
[0676] 144. The method of any one of embodiments 132 to 143,
wherein the proelastase protein is stored at a temperature of
-80.degree. C. to +4.degree. C.
[0677] 145. The method of any one of embodiments 132 to 144,
wherein said activating step comprises trypsin activation.
[0678] 146. The method of any one of embodiments 132 to 144,
wherein said activating step comprises autoactivation.
[0679] 147. The method of any one of embodiments 132 to 146 which
further comprises the step of isolating said mature elastase
protein.
[0680] 148. The method of any one of embodiments 132 to 147,
wherein the mature elastase protein is a mature type I elastase
protein.
[0681] 149. The method of embodiment 148, wherein the mature type I
elastase protein is a human type I mature elastase protein.
[0682] 150. The method of embodiment 148, wherein the mature type I
elastase protein is a porcine type I mature elastase protein.
[0683] 151. A method of producing a mature type I elastase protein
comprising subjecting a recombinant autoactivated type I
proelastase protein to autoactivation conditions, contacting a
recombinant autoactivated type I proelastase protein with a
catalytic amount of elastase, or both, thereby producing a mature
type I elastase protein.
[0684] 152. The method of embodiment 151, wherein said recombinant
autoactivated type I proelastase protein is obtained by or
obtainable by a process comprising: [0685] (a) culturing the host
cell of embodiment 84 or 86 under conditions in which the protein
is expressed; and [0686] (b) recovering the expressed protein,
[0687] thereby producing a recombinant autoactivated type I
proelastase protein.
[0688] 153. The method of embodiment 152, wherein the host cell is
cultured in complex medium.
[0689] 154. The method of embodiment 153, wherein the complex
medium is buffered methanol-complex medium or buffered
glycerol-complex medium.
[0690] 155. The method of any one of embodiments 152 to 154,
wherein the host cell is cultured in the presence of a citrate,
succinate or acetate compound.
[0691] 156. The method of embodiment 155, wherein the citrate,
succinate or acetate compound is sodium citrate, sodium succinate
or sodium acetate, respectively.
[0692] 157. The method of embodiment 155 or embodiment 156, wherein
one citrate, succinate or acetate compound is present in said
culture at a concentration of 5-50 mM, 7.5-100 mM, 10-150 mM,
50-200 mM, 100-150 mM, 75-125 mM, or 90-110 mM.
[0693] 158. The method of embodiment 155 or embodiment 156, wherein
more than one citrate, succinate or acetate compound is present in
said culture, and wherein the total concentration of citrate,
succinate or acetate compounds in said solution is 5-50 mM, 7.5-100
mM, 10-150 mM, 50-200 mM, 100-150 mM, 75-125 mM, or 90-110 mM.
[0694] 159. The method of any one of embodiments 152 to 158,
wherein recovering the expressed protein comprises recovering the
supernatant.
[0695] 160. The method of embodiment 159, wherein said
autoactivation step is performed in the supernatant.
[0696] 161. The method of any one of embodiments 152 to 154,
wherein the expressed protein is recovered from the
supernatant.
[0697] 162. The method of embodiment 161, wherein said
autoactivation step is performed in the supernatant.
[0698] 163. The method of embodiment 161, which further comprises
the step of purifying the expressed protein.
[0699] 164. The method of any one of embodiments 151 to 163,
wherein subjecting the recombinant autoactivated type I proelastase
protein to autoactivation conditions comprises raising the pH of a
solution containing the recovered protein.
[0700] 165. The method of embodiment 164, wherein the pH of the
solution is raised to a basic pH.
[0701] 166. The method of embodiment 165, wherein the basic pH is
in a range from 7 to 9.
[0702] 167. The method of embodiment 166, wherein the basic pH is
8.
[0703] 168. The method of any one of embodiments 164 to 167,
wherein the recovered protein is at a concentration of 10 mg/ml or
less in said solution.
[0704] 169. The method of any one of embodiments 164 to 167,
wherein the recovered protein is at a concentration of 5 mg/ml or
less in said solution.
[0705] 170. The method of any one of embodiments 164 to 167,
wherein the recovered protein is at a concentration of 2 mg/ml or
less in said solution.
[0706] 171. The method of any one of embodiments 164 to 167,
wherein the recovered protein is at a concentration of 1 mg/ml or
less in said solution.
[0707] 172. The method of any one of embodiments 164 to 167,
wherein the recovered protein is at a concentration of 0.5 mg/ml or
less in said solution.
[0708] 173. The method of any one of embodiments 164 to 167,
wherein the recovered protein is at a concentration of 0.25 mg/ml
or less in said solution.
[0709] 174. The method of any one of embodiments 165 to 173,
wherein the recovered protein is at a concentration of at least 0.1
mg/ml in said solution.
[0710] 175. The method of any one of embodiments 165 to 173,
wherein the recovered protein is at a concentration of at least 0.2
mg/ml in said solution.
[0711] 176. The method of any one of embodiments 165 to 175 wherein
the recovered protein is exposed to the basic pH for a period of
0.5 to 8 hours.
[0712] 177. The method of embodiment 174, wherein the recovered
protein is exposed to the basic pH for a period of 2 to 7
hours.
[0713] 178. The method of embodiment 177, wherein the recovered
protein is exposed to the basic pH for a period of 6 hours.
[0714] 179. The method of any one of embodiments 165 to 178,
wherein said exposure to a basic pH is performed at a temperature
of 22.degree. C. to 28.degree. C.
[0715] 180. The method of embodiment 179, wherein said exposure to
a basic pH is performed at a temperature of 26.degree. C.
[0716] 181. The method of embodiment 152 to 180, wherein the
recovered protein is stored prior to autoactivation.
[0717] 182. The method of embodiment 181, wherein the recovered
protein is lyophilized prior to storage.
[0718] 183. The method of embodiment 182, wherein the recovered
protein is purified prior to lyophilization.
[0719] 184. The method of any one of embodiments 181 to 183,
wherein the recovered protein is stored for a period of at least
one day, at least one week, at least one month or at least three
months.
[0720] 185. The method of embodiment 184, wherein the recovered
protein is stored at a temperature from 80.degree. C. to +4.degree.
C.
[0721] 186. The method of any one of embodiments 150 to 185 inasfar
as such embodiments do not depend on embodiments 160 or 162,
wherein the recombinant autoactivated type I proelastase is
purified prior to subjecting it to autoactivation conditions.
[0722] 187. The method of any one of embodiments 150 to 186, which
further comprises the step of isolating said mature type I elastase
protein.
[0723] 188. The method of any one of embodiments 150 to 187,
wherein the mature type I elastase protein is a human type I mature
elastase protein.
[0724] 189. The method of any one of embodiments 150 to 187,
wherein the mature type I elastase protein is a porcine type I
mature elastase protein.
[0725] 190. A method of producing a mature type I elastase protein
comprising: [0726] (a) culturing the host cell of embodiment 84 or
86 under conditions in which the protein is expressed; [0727] (b)
recovering the expressed protein; [0728] (c) purifying the
recovered protein; [0729] (d) raising the pH of a solution
containing the protein or contacting the recovered protein with a
catalytic amount of elastase to produce a mature type I elastase
protein, [0730] thereby producing a mature type I elastase
protein.
[0731] 191. The method of embodiment 190, wherein the host cell is
cultured in complex medium.
[0732] 192. The method of embodiment 191, wherein the complex
medium is buffered methanol-complex medium or buffered
glycerol-complex medium.
[0733] 193. The method of any one of embodiments 190 to 192,
wherein the host cell is cultured in the presence of a citrate,
succinate or acetate compound.
[0734] 194. The method of embodiment 193, wherein the citrate,
succinate or acetate compound is sodium citrate, sodium succinate
or sodium acetate, respectively.
[0735] 195. The method of embodiment 193 or embodiment 194, wherein
one citrate, succinate or acetate compound is present in said
culture at a concentration of 5-50 mM, 7.5-100 mM, 10-150 mM,
50-200 mM, 100-150 mM, 75-125 mM, or 90-110 mM.
[0736] 196. The method of embodiment 193 or embodiment 194, wherein
more than one citrate, succinate or acetate compound is present in
said culture, and wherein the total concentration of citrate,
succinate or acetate compounds in said solution is 5-50 mM, 7.5-100
mM, 10-150 mM, 50-200 mM, 100-150 mM, 75-125 mM, or 90-110 mM.
[0737] 197. The method of any one of embodiments 190 to 196, which
further comprises the step of (e) purifying said mature type I
elastase.
[0738] 198. The method of any one of embodiments 190 to 197,
wherein the mature type I elastase protein is a human type I mature
elastase protein.
[0739] 199. The method of any one of embodiments 190 to 197,
wherein the mature type I elastase protein is a porcine type I
mature elastase protein.
[0740] 200. A method of producing a mature type I elastase protein
comprising: [0741] (a) culturing the host cell of embodiment 84 or
86 under conditions in which the protein is expressed; [0742] (b)
recovering the expressed protein; and [0743] (c) exposing the
recovered protein to a basic pH until a mature protein is
produced;
[0744] thereby producing a mature type I elastase protein.
[0745] 201. The method of embodiment 200, wherein the host cell is
cultured in complex medium.
[0746] 202. The method of embodiment 201, wherein the complex
medium is buffered methanol-complex medium or buffered
glycerol-complex medium.
[0747] 203. The method of any one of embodiments 200 to 202,
wherein the host cell is cultured in the presence of a citrate,
succinate or acetate compound.
[0748] 204. The method of embodiment 203, wherein the citrate,
succinate or acetate compound is sodium citrate, sodium succinate
or sodium acetate, respectively.
[0749] 205. The method of embodiment 203 or embodiment 204, wherein
one citrate, succinate or acetate compound is present in said
culture at a concentration of 5-50 mM, 7.5-100 mM, 10-150 mM,
50-200 mM, 100-150 mM, 75-125 mM, or 90-110 mM.
[0750] 206. The method of embodiment 203 or embodiment 204, wherein
more than one citrate, succinate or acetate compound is present in
said culture, and wherein the total concentration of citrate,
succinate or acetate compounds in said solution is 5-50 mM, 7.5-100
mM, 10-150 mM, 50-200 mM, 100-150 mM, 75-125 mM, or 90-110 mM.
[0751] 207. The method of any one of embodiments 200 to 206,
wherein the basic pH is 7 to 9.
[0752] 208. The method of embodiment 207, wherein the basic pH is
8.
[0753] 209. The method of any one of embodiments 200 to 208,
wherein recovered protein is at a concentration of 10 mg/ml or less
when exposed to the basic pH.
[0754] 210. The method of any one of embodiments 200 to 208,
wherein recovered protein is at a concentration of 5 mg/ml or less
when exposed to the basic pH.
[0755] 211. The method of any one of embodiments 200 to 208,
wherein recovered protein is at a concentration of 2 mg/ml or less
when exposed to the basic pH.
[0756] 212. The method of any one of embodiments 200 to 208,
wherein recovered protein is at a concentration of 1 mg/ml or less
when exposed to the basic pH.
[0757] 213. The method of any one of embodiments 200 to 208,
wherein recovered protein is at a concentration of 0.5 mg/ml or
less when exposed to the basic pH.
[0758] 214. The method of any one of embodiments 200 to 208,
wherein recovered protein is at a concentration of 0.25 mg/ml or
less when exposed to the basic pH.
[0759] 215. The method of any one of embodiments 209 to 214,
wherein recovered protein is at a concentration of at least 0.1
mg/ml when exposed to the basic pH.
[0760] 216. The method of any one of embodiments 209 to 214,
wherein recovered protein is at a concentration of at least 0.2
mg/ml when exposed to the basic pH.
[0761] 217. The method of any one of embodiments 200 to 214,
wherein the recovered protein is exposed to the basic pH for a
period of 0.5 to 8 hours.
[0762] 218. The method of embodiment 217, wherein the recovered
protein is exposed to the basic pH for a period of 2 to 7
hours.
[0763] 219. The method of embodiment 218, wherein the recovered
protein is exposed to the basic pH for a period of 6 hours.
[0764] 220. The method of any one of embodiments 200 to 219,
wherein said exposure to a basic pH is performed at a temperature
of 22.degree. C. to 28.degree. C.
[0765] 221. The method of embodiment 220, wherein said exposure to
a basic pH is performed at a temperature of 26.degree. C.
[0766] 222. The method of any one of embodiments 200 to 221, which
further comprises the step of (d) isolating said mature type I
elastase protein.
[0767] 223. The method of any one of embodiments 150 to 222,
wherein the mature type I elastase is a mature porcine type I
elastase.
[0768] 224. The method of any one of embodiments 150 to 222,
wherein the mature type I elastase is a mature human type I
elastase.
[0769] 225. A method of producing a formulation of mature elastase
protein, comprising: [0770] (a) subjecting an autoactivated
proelastase protein to autoactivating conditions to produce mature
elastase protein; and [0771] (b) formulating the mature elastase
protein,
[0772] thereby producing a formulation of mature elastase
protein.
[0773] 226. The method of embodiment 225, wherein the autoactivated
proelastase protein is recombinant.
[0774] 227. The method of embodiment 226 further comprising, prior
to step (a) recovering the proelastase protein from a culture of a
host cell capable of expressing said autoactivated proelastase
protein grown under conditions in which the proelastase protein is
expressed.
[0775] 228. The method of embodiment 225 or embodiment 226, further
comprising, prior to step (b), purifying said mature elastase
protein.
[0776] 229. The method of any one of embodiments 225 to 228,
wherein said formulating step comprises lyophilizing said mature
elastase protein.
[0777] 230. The method of embodiment 229, wherein the mature
elastase protein is not mixed with buffer or buffer ingredients
prior to lyophilization.
[0778] 231. The method of embodiment 230, wherein the mature
elastase protein is mixed with one or more buffer ingredients
following lyophilization.
[0779] 232. The method of embodiment 229, wherein the mature
elastase protein is mixed with buffer or one or more buffer
ingredients prior to lyophilization.
[0780] 233. The method of embodiment 231 or 232, wherein the buffer
is a phosphate buffered saline ("PBS") buffer or the buffer
ingredients are PBS buffer ingredients.
[0781] 234. The method of any one of embodiments 231 to 233,
wherein the buffer comprises dextran or wherein the buffer
ingredients comprise dextran.
[0782] 235. The method of embodiment 234, wherein the dextran is
dextran-18.
[0783] 236. The method of any one of embodiments 230 to 235,
wherein the buffer comprises polysorbate 80.
[0784] 237. The method of any one of embodiments 229 to 236,
wherein said formulating step further comprises reconstituting the
lyophilized mature elastase protein with a liquid.
[0785] 238. The method of embodiment 237, wherein the liquid is
water.
[0786] 239. The method of embodiment 237, wherein the liquid is a
buffer.
[0787] 240. The method of embodiment 239, wherein the buffer is
full strength buffer, greater than full strength buffer, or less
than full strength buffer.
[0788] 241. The method of any one of embodiments 237 to 240,
wherein upon reconstitution a solution of mature elastase protein
in full strength buffer, greater than full strength buffer, or less
than full strength buffer is produced.
[0789] 242. The method of embodiment 241, wherein buffer
ingredients are present in the lyophilisate, added upon
reconstitution, or both.
[0790] 243. The method of any one of embodiments 240 to 242,
wherein full strength buffer comprises 1.times.PBS.
[0791] 244. The method of embodiment 240 or 241, wherein the less
than full strength buffer comprises 0.1.times.PBS to
0.5.times.PBS.
[0792] 245. The method of embodiment 244, wherein the less than
full strength buffer comprises 0.1.times.PBS.
[0793] 246. The method of embodiment 244, wherein the less than
full strength buffer comprises 0.5.times.PBS.
[0794] 247. The method of embodiment 240 or 241, wherein the
greater than full strength buffer comprises 1. X PBS to
3.times.PBS.
[0795] 248. The method of embodiment 247, wherein the greater than
full strength buffer comprises 1.5.times.PBS to 2.times.PBS.
[0796] 249. The method of any one of embodiments 241 to 248,
wherein the buffer comprises dextran.
[0797] 250. The method of embodiment 249, wherein the dextran is
dextran 18.
[0798] 251. The method of any one of embodiments 241 to 250,
wherein the buffer comprises polysorbate 80.
[0799] 252. The method of any one of embodiments 241 to 251,
wherein the buffer is at a pH of 7 to 8.
[0800] 253. The method of embodiment 252, wherein the buffer is at
a pH of 7.4.
[0801] 254. The method of any one of embodiments 237 to 253,
wherein the mature elastase protein is reconstituted to a
concentration of 0.001 mg/ml to 50 mg/ml.
[0802] 255. The method of embodiment 254, wherein the mature
elastase protein is reconstituted to a concentration of 0.1 mg/ml
to 40 mg/ml.
[0803] 256. The method of embodiment 255, wherein the mature
elastase protein is reconstituted to a concentration of 5 mg/ml to
30 mg/ml.
[0804] 257. The method of embodiment 256, wherein the mature
elastase protein is reconstituted to a concentration of 10 mg/ml to
20 mg/ml.
[0805] 258. The method of any one of embodiments 225 to 257,
wherein said formulation is a pharmaceutical composition comprising
said mature elastase protein.
[0806] 259. The method of any one of embodiments 225 to 258,
wherein the mature type I elastase protein is a human type I mature
elastase protein.
[0807] 260. The method of any one of embodiments 225 to 258,
wherein the mature type I elastase protein is a porcine type I
mature elastase protein.
[0808] 261. A method of producing a lyophilized mature type I
elastase comprising: [0809] (a) producing a mature type I elastase
according to the method of any one of embodiments 151 to 181;
[0810] (b) isolating mature type I elastase; and [0811] (c)
lyophilizing said isolated mature type I elastase,
[0812] thereby producing a lyophilized mature type I elastase.
[0813] 262. The method of embodiment 261, wherein the lyophilized
mature type I elastase is 95% to 100% pure.
[0814] 263. The method of embodiment 261 or embodiment 262, wherein
the lyophilized mature type I elastase is at least 95% pure.
[0815] 264. The method of embodiment 261 or embodiment 262, wherein
the lyophilized mature type I elastase is at least 98% pure.
[0816] 265. The method of any one of embodiments 261 to 264,
wherein the lyophilized mature type I elastase is purified to
homogeneity.
[0817] 266. The method of any one of embodiments 261 to 265,
wherein the mature type I elastase protein is a human type I mature
elastase protein.
[0818] 267. The method of any one of embodiments 261 to 265,
wherein the mature type I elastase protein is a porcine type I
mature elastase protein.
[0819] 268. The method of any one of embodiments 130, 149, 188,
198, 223, 259, and 267, wherein the mature human type I elastase
protein consists essentially of SEQ ID NO:5, SEQ ID NO:84, or SEQ
ID NO:87.
[0820] 269. The method of embodiment 268, wherein the mature human
type I elastase protein consists essentially of SEQ ID NO:1 or SEQ
ID NO:4.
[0821] 270. The method of any one of embodiments 131, 150, 189,
199, 224, 260, and 268, wherein the mature porcine type I elastase
protein consists essentially of SEQ ID NO:39.
[0822] 271. The method of any one of embodiments 111 to 128, 132 to
148, 151 to 197 and 225 to 257, wherein the proelastase protein is
a protein of any one of embodiments 13 to 27.
[0823] 272. The method of any one of embodiments 200 to 222,
wherein the expressed protein is a protein of any one of
embodiments 13 to 27.
[0824] 273. A mature human type I elastase produced by or
obtainable by the method of any one of embodiments 149, 188, 198,
and 223.
[0825] 274. The mature human type I elastase of embodiment 273
which has a specific activity of 1 to 40 U/mg protein.
[0826] 275. A mature porcine type I elastase produced by or
obtainable by the method of any one of embodiments 150, 189, 199,
and 224.
[0827] 276. The mature human type I elastase of embodiment 275
which has a specific activity of 10 to 100 U/mg protein.
[0828] 277. A pharmaceutical composition comprising a
therapeutically effective amount of (a) the mature human type I
elastase of embodiment 273 or embodiment 274 or (b) a formulation
of mature human type I elastase produced by or obtainable by the
method of embodiment 259.
[0829] 278. The pharmaceutical composition of embodiment 277,
wherein the mature human type I elastase protein consists
essentially of SEQ ID NO:5, SEQ ID NO:84, or SEQ ID NO:87.
[0830] 279. The pharmaceutical composition of embodiment 278,
wherein the mature human type I elastase protein consists
essentially of SEQ ID NO:1 or SEQ ID NO:4.
[0831] 280. A pharmaceutical composition comprising a
therapeutically effective amount of (a) the mature porcine type I
elastase of embodiment 275 or embodiment 276 or (b) a formulation
of mature porcine type I elastase produced by or obtainable by the
method of embodiment 260.
[0832] 281. The pharmaceutical composition of embodiment 280,
wherein the mature porcine type I elastase protein consists
essentially of SEQ ID NO:39.
[0833] 282. A pharmaceutical composition comprising (i) a
therapeutically effective amount of mature human type I elastase
and (ii) a pharmaceutically acceptable carrier, which
pharmaceutical composition is characterized by at least one of the
following properties: [0834] (a) the composition is free of
trypsin; [0835] (b) the composition is substantially free of
trypsin; [0836] (c) the composition is free of any protein
consisting of SEQ ID NOS: 2, 3, 37, 38, 70 and/or 71; [0837] (d)
the composition is substantially free of any protein consisting of
SEQ ID NOS: 2, 3, 37, 38, 70 and/or 71; [0838] (e) the composition
is free of bacterial proteins; [0839] (f) the composition is
substantially free of bacterial proteins; [0840] (g) the
composition is free of mammalian proteins other than said mature
human type I elastase; [0841] (h) the composition is substantially
free of mammalian proteins other than said mature human type I
elastase.
[0842] 283. A pharmaceutical composition comprising (i) a
therapeutically effective amount of mature human type I elastase
and (ii) a pharmaceutically acceptable carrier, which
pharmaceutical composition is characterized by at least one of the
following properties: [0843] (a) the composition is free of
trypsin; [0844] (b) the composition is substantially free of
trypsin; [0845] (c) the composition is free of any protein
consisting of SEQ ID NOS:70 and 71; [0846] (d) the composition is
substantially free of any protein consisting of SEQ ID NOS:2 and 3;
[0847] (e) the composition is free of bacterial proteins; [0848]
(f) the composition is substantially free of bacterial proteins;
[0849] (g) the composition is free of mammalian proteins other than
said mature human type I elastase; [0850] (h) the composition is
substantially free of mammalian proteins other than said mature
human type I elastase.
[0851] 284. The pharmaceutical composition of embodiment 282 or
embodiment 283 which is characterized by at least two of the
properties (a) to (h).
[0852] 285. The pharmaceutical composition of embodiment 284,
wherein said at least two properties include (a) and (c).
[0853] 286. The pharmaceutical composition of embodiment 284,
wherein said at least two properties include (b) and (d).
[0854] 287. The pharmaceutical composition of embodiment 282 or
embodiment 283 which is characterized by at least three of the
properties (a) to (h).
[0855] 288. The pharmaceutical composition of embodiment 284,
wherein said at least three properties include (a), (c) and
(e).
[0856] 289. The pharmaceutical composition of embodiment 284,
wherein said at least three properties include (b), (d), and
(f).
[0857] 290. The pharmaceutical composition of embodiment 282 or
embodiment 283 which is characterized by at least four of the
properties (a) to (h).
[0858] 291. The pharmaceutical composition of embodiment 284,
wherein said at least four properties include (a), (c), (e) and
(g).
[0859] 292. The pharmaceutical composition of embodiment 284,
wherein said at least four properties include (b), (d), (f), and
(h).
[0860] 293. The pharmaceutical composition of embodiment 282 or
embodiment 283 which is characterized by at least five of the
properties (a) to (h).
[0861] 294. The pharmaceutical composition of embodiment 282 or
embodiment 283 which is characterized by at least six of the
properties (a) to (h).
[0862] 295. The pharmaceutical composition of embodiment 282 or
embodiment 283 which is characterized by at least seven of the
properties (a) to (h).
[0863] 296. The pharmaceutical composition of embodiment 282 or
embodiment 283 which is characterized by all properties (a) to
(h).
[0864] 297. The pharmaceutical composition of any one of
embodiments 282 to 296 which is free or substantially free of one,
two, three or all four proteins consisting of SEQ ID NO:85, 86, 94
and 95.
[0865] 298. The pharmaceutical composition of any one of
embodiments 282 to 297 which is free or substantially free of
proteins consisting of SEQ ID NO:85 and SEQ ID NO:86.
[0866] 299. The pharmaceutical composition of any one of
embodiments 282 to 297 which is free or substantially free of
proteins consisting of SEQ ID NO:94 and SEQ ID NO:95.
[0867] 300. The pharmaceutical composition of any one of
embodiments 282 to 297 which is free or substantially free of one,
two, or all three proteins consisting of SEQ ID NO:106, 107 and
108.
[0868] 301. The pharmaceutical composition of any one of
embodiments 282 to 300 which contains pharmaceutically acceptable
levels of endotoxins.
[0869] 302. The pharmaceutical composition of embodiment 301,
wherein said pharmaceutical composition is a liquid composition and
wherein said pharmaceutically acceptable levels of endotoxins are 8
EU/ml or less.
[0870] 303. The pharmaceutical composition of embodiment 302,
wherein said pharmaceutically acceptable levels of endotoxins are 5
EU/ml or less.
[0871] 304. The pharmaceutical composition of embodiment 301,
wherein said pharmaceutical composition is a solid composition and
wherein said pharmaceutically acceptable levels of endotoxins are
10 EU or less per gram of mature human type I elastase.
[0872] 305. The pharmaceutical composition of embodiment 304
wherein said pharmaceutically acceptable levels of endotoxins are 5
EU or less per gram of mature human type I elastase.
[0873] 306. The pharmaceutical composition of any one of
embodiments 282 to 305 in which the mature human type I elastase is
characterized by a specific activity of 1 to 40 U/mg of
protein.
[0874] 307. The pharmaceutical composition of embodiment 306 in
which the mature human type I elastase is characterized by a
specific activity of 25 to 35 U/mg of protein.
[0875] 308. The pharmaceutical composition of embodiment 306 in
which the mature human type I elastase is characterized by a
specific activity of greater than 10 U/mg of protein.
[0876] 309. The pharmaceutical composition of embodiment 306 in
which the mature human type I elastase is characterized by a
specific activity of greater than 20 U/mg of protein.
[0877] 310. The pharmaceutical composition of any one of
embodiments 282 to 309, wherein the mature human type I elastase
consists essentially of SEQ ID NO: 5, 84, or 87.
[0878] 311. The pharmaceutical composition of embodiment 310,
wherein the mature human type I elastase consists essentially of
SEQ ID NO:1 or SEQ ID NO:4.
[0879] 312. The pharmaceutical composition of any one of
embodiments 282 to 311 in which the trypsin activity corresponds to
less than 4 ng per 1 mg of mature human type I elastase
protein.
[0880] 313. The pharmaceutical composition of embodiment 312 in
which the trypsin activity corresponds to less than 2 ng per 1 mg
of mature human type I elastase protein.
[0881] 314. A lyophilized formulation of a protein consisting
essentially of SEQ ID NO: 5, 84, or 87 which upon reconstitution to
a concentration of said protein of 1 mg/ml the trypsin activity
corresponds to less than 2 ng/ml trypsin.
[0882] 315. The lyophilized formulation of embodiment 314, wherein
the moisture content is less than 5%.
[0883] 316. The lyophilized formulation of embodiment 315 which
comprises sodium ions, potassium ions, phosphate ions, and chloride
ions.
[0884] 317. The lyophilized formulation of embodiment 315 which
comprises polysorbate-80.
[0885] 318. The lyophilized formulation of embodiment 315 which
comprises dextran.
[0886] 319. The lyophilized formulation of embodiment 318, wherein
the dextran is dextran-18.
[0887] 320. The lyophilized formulation of embodiment 311 which
comprises sodium ions, potassium ions, phosphate ions, chloride
ions and polysorbate-80.
[0888] 321. The lyophilized formulation of embodiment 314 which
comprises sodium ions, potassium ions, phosphate ions, chloride
ions and dextran.
[0889] 322. The lyophilized formulation of embodiment 321, wherein
the dextran is dextran-18.
[0890] 323. The lyophilized formulation of embodiment 314 which
comprises sodium ions, potassium ions, phosphate ions, chloride
ions, polysorbate-80, and dextran.
[0891] 324. The lyophilized formulation of embodiment 323, wherein
the dextran is dextran-18.
[0892] 325. A liquid formulation comprising a solution of a least
0.1 mg/ml of a protein consisting essentially of SEQ ID NO: 5, 84,
or 87 in which the trypsin activity corresponds to less than 2
ng/ml trypsin.
[0893] 326. The liquid formulation of embodiment 325, wherein the
solution is a buffered solution.
[0894] 327. The liquid formulation of embodiment 326, wherein the
solution is buffered to a pH of 7 to 8.
[0895] 328. The liquid formulation of embodiment 326 in which the
solution is PBS.
[0896] 329. The liquid formulation of embodiment 328 in which the
solution comprises 137 mM sodium chloride, 2.7 mM potassium
phosphate, and 10 mM sodium phosphate.
[0897] 330. The liquid formulation of any one of embodiments 325 to
329 which further comprises one or more excipients.
[0898] 331. The liquid formulation of embodiment 330 in which said
one or more excipients comprises dextran-18.
[0899] 332. The liquid formulation of embodiment 331 in which the
dextran-18 is in the amount of 8% weight/volume of said
solution.
[0900] 333. The liquid formulation of embodiment 330 in which said
one or more excipients comprises polysorbate-80.
[0901] 334. The liquid formulation of embodiment 333 in which the
polysorbate-80 is in the amount of 0.01% weight/volume of said
solution.
[0902] 335. The liquid formulation of any one of embodiments 325 to
334 in which the concentration of said protein in said solution is
in the range of 0.001 mg/ml to 40 mg/ml.
[0903] 336. The liquid formulation of embodiment 335 in which the
concentration of said protein in said solution is in the range of
0.1 mg/ml to 20 mg/ml.
[0904] 337. The liquid formulation of any one of embodiments 325 to
336 with further comprises one or more of a preservative, a
solubilizer and a coloring agent.
[0905] 338. The liquid formulation of any one of embodiments 325 to
337 which is substantially free of mammalian proteins other than
said protein of SEQ ID NO:5, 84 or 87.
[0906] 339. The liquid formulation of any one of embodiments 325 to
338 which is substantially free of bacterial proteins.
[0907] 340. A formulation made by or obtainable by the method of
any one of embodiments 225 to 258.
[0908] 341. The formulation of embodiment 340 which is a liquid
formulation.
[0909] 342. The formulation of embodiment 340 or 341 in which the
mature type I elastase protein is a human type I mature elastase
protein.
[0910] 343. The formulation of embodiment 342, wherein the mature
human type I elastase protein consists essentially of SEQ ID NO:5,
SEQ ID NO:84, or SEQ ID NO:87.
[0911] 344. The formulation of embodiment 342, wherein the mature
human type I elastase protein consists essentially of SEQ ID NO:1
or SEQ ID NO:4.
[0912] 345. The formulation of embodiment 340 or 341 in which the
mature type I elastase protein is a porcine type I mature elastase
protein.
[0913] 346. The formulation of embodiment 345, wherein the mature
porcine type I elastase protein consists essentially of SEQ ID
NO:39.
[0914] 347. A method of removing one or more incorrectly processed
mature elastase proteins from a mixture of correctly and
incorrectly processed mature elastase proteins, said method
comprising: [0915] (a) subjecting a composition comprising a
mixture of correctly and incorrectly processed mature elastase
proteins to a pH at which the correctly processed mature enzyme is
active; [0916] (b) maintaining such pH until such time that one or
more incorrectly processed mature elastase proteins are degraded,
thereby removing said one or more incorrectly processed mature
elastase proteins from a mixture of correctly and incorrectly
processed mature elastase proteins.
[0917] 348. The method of embodiment 347, wherein said one or more
incorrectly processed mature elastase proteins contain at least one
additional or fewer amino acid at the N-terminus relative to
correctly processed mature elastase proteins.
[0918] 349. The method of embodiment 347 or 348 wherein said pH is
between 5 and 12.
[0919] 350. The method of any one of embodiments 347 to 349,
wherein said one or more incorrectly processed mature elastase
proteins are degraded by 50% to 100%.
[0920] 351. The method of embodiment 348, wherein said one or more
incorrectly processed mature elastase proteins are degraded by 50%
to 99%.
[0921] 352. The method of embodiment 348, wherein said one or more
incorrectly processed mature elastase proteins are degraded by 50%
to 98%.
[0922] 353. The method of embodiment 348, wherein said one or more
incorrectly processed mature elastase proteins are degraded by 50%
to 95%.
[0923] 354. The method of embodiment 348, wherein said one or more
incorrectly processed mature elastase proteins are degraded by 50%
to 90%.
[0924] 355. The method of any one of embodiments 350 to 354,
wherein more than one incorrectly processed mature elastase protein
is degraded by at least 50%.
[0925] 356. The method of embodiment 355, wherein all incorrectly
processed mature elastase proteins in the mixture are degraded by
at least 50%.
[0926] 357. The method of any one of embodiments 350 to 354 wherein
at least one incorrectly processed mature elastase protein is
degraded by at least 75%.
[0927] 358. The method of embodiment 357, wherein more than one
incorrectly processed mature elastase protein is degraded by at
least 75%.
[0928] 359. The method of embodiment 357, wherein all incorrectly
processed mature elastase proteins in the mixture are degraded by
at least 75%.
[0929] 360. The method of any one of embodiments 350 to 354 wherein
at least one incorrectly processed mature elastase protein is
degraded by at least 90%.
[0930] 361. The method of embodiment 360, wherein more than one
incorrectly processed mature elastase protein is degraded by at
least 90%.
[0931] 362. The method of embodiment 360, wherein all incorrectly
processed mature elastase proteins in the mixture are degraded by
at least 90%.
[0932] 363. The method of any one of embodiments 347 to 362,
wherein the composition is a liquid composition in which the total
elastase protein is at a concentration of 10 mg/ml or less.
[0933] 364. The method of any one of embodiments 347 to 362,
wherein the composition is a liquid composition in which the total
elastase protein is at a concentration of 5 mg/ml or less.
[0934] 365. The method of any one of embodiments 347 to 362,
wherein the composition is a liquid composition in which the total
elastase protein is at a concentration of 2 mg/ml or less.
[0935] 366. The method of any one of embodiments 347 to 362,
wherein the composition is a liquid composition in which the total
elastase protein is at a concentration of 1 mg/ml or less.
[0936] 367. The method of any one of embodiments 347 to 362,
wherein the composition is a liquid composition in which the total
elastase protein is at a concentration of 0.5 mg/ml or less.
[0937] 368. The method of any one of embodiments 347 to 362,
wherein the composition is a liquid composition in which the total
elastase protein is at a concentration of 0.25 mg/ml or less.
[0938] 369. The method of any one of embodiments 363 to 368,
wherein the composition is a liquid composition in which the total
elastase protein is at a concentration of at least 0.1 mg/ml.
[0939] 370. The method of any one of embodiments 363 to 368,
wherein the composition is a liquid composition in which the total
elastase protein is at a concentration of at least 0.2 mg/ml.
[0940] 371. The method of any one of embodiments 347 to 370,
wherein the trypsin activity in said composition is less than 4
ng/ml trypsin per mg of total elastase proteins.
[0941] 372. The method of any one of embodiments 347 to 370,
wherein the trypsin activity in said composition is less than 2
ng/ml trypsin per mg of total elastase proteins.
[0942] 373. The method of any one of embodiments 347 to 372,
wherein the composition is free or substantially free of a protein
consisting of SEQ ID NO:104 and/or is free or substantially free of
a protein consisting of SEQ ID NO:105.
[0943] 374. A method of producing a pharmaceutical composition
comprising a mature type I elastase, said method comprising (i)
producing a lyophilized mature type I elastase by the method of any
one of embodiments 261 to 265; and (ii) reconstituting the
lyophilized mature type I elastase in water or a pharmaceutically
acceptable carrier, thereby producing a pharmaceutical composition
comprising a mature human type I elastase.
[0944] 375. A method of producing a pharmaceutical composition
comprising a mature type I elastase, said method comprising
reconstituting the lyophilized formulation of any one of
embodiments 314 to 324 in water or a pharmaceutically acceptable
carrier, thereby producing a pharmaceutical composition comprising
a mature type I elastase.
[0945] 376. A method of embodiment 374 or embodiment 375 where the
pharmaceutical composition comprises phosphate.
[0946] 377. The method of any one of embodiments 374 to 376,
wherein the mature type I elastase is mature human type I
elastase.
[0947] 378. The method of embodiment 377 characterized by a
specific activity of 1 to 40 U/mg protein.
[0948] 379. The method of embodiment 377 wherein the mature type I
elastase is characterized by a specific activity of 25 to 35 U/mg
protein.
[0949] 380. The method of any one of embodiments 377 to 379,
wherein the mature human type I elastase in said pharmaceutical
composition maintains 60% to 100% of its specific activity after at
least a week of storage at 4.degree. C., after at least a month of
storage at 4.degree. C., after at least two months of storage at
4.degree. C., after at least three months of storage at 4.degree.
C., or after at least month six months of storage at 4.degree.
C.
[0950] 381. The method of any one of embodiments 377 to 379,
wherein the mature human type I elastase in said pharmaceutical
composition maintains 60% to 98% of its specific activity after at
least a week of storage at 4.degree. C., after at least a month of
storage at 4.degree. C., after at least two months of storage at
4.degree. C., after at least three months of storage at 4.degree.
C., or after at least month six months of storage at 4.degree.
C.
[0951] 382. The method of any one of embodiments 377 to 379,
wherein the mature human type I elastase in said pharmaceutical
composition maintains 60% to 95% of its specific activity after at
least a week of storage at 4.degree. C., after at least a month of
storage at 4.degree. C., after at least two months of storage at
4.degree. C., after at least three months of storage at 4.degree.
C., or after at least month six months of storage at 4.degree.
C.
[0952] 383. The method of any one of embodiments 377 to 379,
wherein the mature human type I elastase in said pharmaceutical
composition maintains 60% to 90% of its specific activity after at
least a week of storage at 4.degree. C., after at least a month of
storage at 4.degree. C., after at least two months of storage at
4.degree. C., after at least three months of storage at 4.degree.
C., or after at least month six months of storage at 4.degree.
C.
[0953] 384. The method of any one of embodiments 377 to 379,
wherein the mature human type I elastase in said pharmaceutical
composition maintains 60% to 80% of its specific activity after at
least a week of storage at 4.degree. C., after at least a month of
storage at 4.degree. C., after at least two months of storage at
4.degree. C., after at least three months of storage at 4.degree.
C., or after at least month six months of storage at 4.degree.
C.
[0954] 385. The method of any one of embodiments 377 to 384,
wherein the mature human type I elastase in said pharmaceutical
composition maintains at least 70% of its specific activity after a
week of storage at 4.degree. C.
[0955] 386. A pharmaceutical composition produced by or obtainable
by the method of any one of embodiments to 374 to 385.
[0956] 387. A method for therapeutically increasing the diameter of
an artery or vein in a human subject in need thereof, the method
comprising: locally administering to the wall of the artery or vein
in the human subject (a) the pharmaceutical composition of any one
of embodiments 277 to 313 and 386, (b) the liquid formulation of
any one of embodiments 325 to 339, or (c) the formulation of
embodiment 341 in a dose sufficient to increase the diameter of the
artery or vein.
[0957] 388. The method of embodiment 387, wherein the diameter of
the vessel, the lumenal diameter of the vessel, or both, are
increased.
[0958] 389. A method for preventing or treating vasospasm of an
artery or vein in a human subject in need thereof, the method
comprising: locally administering to the wall of the artery or vein
in the human subject (a) the pharmaceutical composition of any one
of embodiments 277 to 313 and 386, (b) the liquid formulation of
any one of embodiments 325 to 339, or (c) the formulation of
embodiment 341 in a dose sufficient to prevent or treat vasospasm
of the artery or vein.
[0959] 390. A method for treating an obstructed artery or vein in a
human subject in need of such treatment, the method comprising:
locally administering to the wall of the artery or vein in the
human subject (a) the pharmaceutical composition of any one of
embodiments 277 to 313 and 386, (b) the liquid formulation of any
one of embodiments 325 to 339, or (c) the formulation of embodiment
341, wherein said administration results in proteolysis of elastin
in the wall of the artery or vein leading to enlargement of the
diameter of the artery or vein.
[0960] 391. A method for treating an artery or vein connected to an
arteriovenous hemodialysis graft or arteriovenous fistula in a
human subject in need of such treatment, the method comprising:
locally administering to the wall of the artery or vein in the
human subject ((a) the pharmaceutical composition of any one of
embodiments 277 to 313 and 386, (b) the liquid formulation of any
one of embodiments 325 to 339, or (c) the formulation of embodiment
341, wherein said administration results in proteolysis of elastin
in the wall of the artery or vein leading to enlargement of the
diameter of the artery or vein.
[0961] 392. A method for treating a vein in a human subject for use
in hemodialysis, the method comprising: locally administering to
the wall of the vein in the human subject (a) the pharmaceutical
composition of any one of embodiments 277 to 313 and 386, (b) the
liquid formulation of any one of embodiments 325 to 339, or (c) the
formulation of embodiment 341, wherein said administration results
in proteolysis of elastin in the wall of the vein leading to
enlargement of the diameter of the vein.
[0962] 393. The method of any one of embodiments 387 to 392, which
further comprises inserting a portion of a delivery apparatus into
the wall of the artery or vein to deliver elastase to the wall of
the artery or vein.
[0963] 394. The method of any one of embodiments 387 to 393,
wherein the pharmaceutical composition or liquid formulation is
administered by a catheter.
[0964] 395. The method of any one of embodiments 387 to 394,
wherein the wherein the pharmaceutical composition or liquid
formulation is administered directly into the wall of the artery or
vein.
[0965] 396. The method of any one of embodiments 387 to 395,
wherein the artery or vein is obstructed.
[0966] 397. The method of embodiment 396, wherein the artery or
vein is obstructed by stenosis.
[0967] 398. The method of embodiment 397, wherein the obstruction
permits passage of an insufficient volume of blood prior to the
treatment.
[0968] 399. The method of embodiment 398, wherein the obstruction
is a stenosis.
[0969] 400. The method of embodiment 399, wherein the artery or
vein is obstructed by intimal hyperplasia.
[0970] 401. The method of any one of embodiments 387 to 400,
wherein the pharmaceutical composition or liquid formulation is
administered to an obstructed coronary or peripheral artery.
[0971] 402. The method of any one of embodiments 387 to 395,
wherein the artery or vein is susceptible to obstruction by intimal
hyperplasia.
[0972] 403. The method of any one of embodiments 387 to 400 and
402, wherein the composition is administered to the wall of a
vein.
[0973] 404. The method of embodiment 403, wherein the vein is
connected to an arteriovenous hemodialysis graft or arteriovenous
fistula.
[0974] 405. The method of embodiment 404, wherein the vein is for
use in hemodialysis.
[0975] 406. The method of embodiment 405, further comprising
directly connecting the vein to an artery or connecting the vein to
an artery via a graft.
[0976] 407. The method of any one of embodiments 387 to 394,
wherein the composition is administered to the adventitial surface
of a surgically exposed artery or vein.
[0977] 408. The method of any one of embodiments 387 to 407,
wherein the mature type I elastase protein in said pharmaceutical
composition, liquid formulation or formulation, respectively, is a
human type I mature elastase protein.
[0978] 409. The method of embodiment 408, wherein the mature human
type I elastase protein consists essentially of SEQ ID NO:5, SEQ ID
NO:84, or SEQ ID NO:87.
[0979] 410. The method of embodiment 408, wherein the mature human
type I elastase protein consists essentially of SEQ ID NO:1 or SEQ
ID NO:4.
[0980] 411. The method of any one of embodiments 387 to 407,
wherein the mature type I elastase protein in said pharmaceutical
composition, liquid formulation or formulation, respectively, is a
porcine type I mature elastase protein.
[0981] 412. The method of embodiment 411, wherein the mature
porcine type I elastase protein consists essentially of SEQ ID
NO:39.
[0982] 413. A unit dosage comprising 0.0033 mg to 200 mg of (a)
mature human type I elastase of embodiment 273 or embodiment 274 or
(b) a formulation of mature human type I elastase produced by or
obtainable by the method of embodiment 259.
[0983] 414. The unit dosage of embodiment 413, wherein the mature
human type I elastase protein consists essentially of SEQ ID NO:5,
SEQ ID NO:84, or SEQ ID NO:87.
[0984] 415. A unit dosage comprising 0.0033 mg to 200 mg of (a) the
mature porcine type I elastase of embodiment 275 or embodiment 276
or (b) a formulation of mature porcine type I elastase produced by
or obtainable by the method of embodiment 260.
[0985] 416. The unit dosage of embodiment 415, wherein the mature
porcine type I elastase protein consists essentially of SEQ ID
NO:39.
[0986] 417. The unit dosage of any one of embodiments 413 to 416
which comprises 0.5 mg to 50 mg of said mature type I elastase.
[0987] 418. The unit dosage of embodiment 417 which comprises 1 mg
to 20 mg of said mature type I elastase.
[0988] 419. The unit dosage of embodiment 418 which comprises 5 mg
to 10 mg of said mature type I elastase.
[0989] 420. The unit dosage of any one of embodiments 413 to 419
which is in a container, pack, dispenser, or catheter.
[0990] 421. A kit comprising an elastase protein according to any
one of embodiments 1 to 39 and 68 to 69 or obtained or obtainable
by the method of any one of embodiments 89 to 224, 261 to 276, and
347 to 373, a nucleic acid according to any one of embodiments 40
to 67, a vector according to any one of embodiments 70 to 72, a
cell according to any one of embodiments 73 to 87, a cell culture
supernatant according to embodiment 88, an elastase formulation
according to any one of embodiments 314 to 346 or obtained or
obtainable by the method of any one of embodiments 261 to 276, a
pharmaceutical composition according to the method of any one of
embodiments 277 to 313 and 386, or obtained or obtainable by the
method of any one of embodiments 374 to 385, or a unit dosage
according to any one of embodiments 415 to 420.
[0991] 422. The kit of embodiment 421 which is a therapeutic
kit.
[0992] 423. The kit of embodiment 422 which comprises a container,
pack, dispenser, or catheter.
[0993] 424. The kit of embodiment 423 which is a manufacturing
kit.
[0994] The invention is further exemplified by the following
Specific Embodiments that pertain to proelastase proteins of SEQ ID
NO:64 and SEQ ID NO:69:
[0995] 1. A protein comprising the amino acid sequence of SEQ ID
NO:64 or SEQ ID NO:69.
[0996] 2. The protein of Specific Embodiment 1 which is
isolated.
[0997] 3. A nucleic acid molecule comprising a nucleotide sequence
encoding a protein of Specific Embodiment 1.
[0998] 4. The nucleic acid molecule of Specific Embodiment 3
wherein the protein comprises a signal sequence operably linked to
said amino acid sequence of SEQ ID NO:64 or SEQ ID NO:69.
[0999] 5. The nucleic acid molecule of Specific Embodiment 4,
wherein the signal sequence is operable in Pichia pastoris.
[1000] 6. The nucleic acid molecule of Specific Embodiment 5,
wherein the signal sequence is a yeast .alpha.-factor signal
peptide.
[1001] 7. A vector comprising the nucleic acid molecule of Specific
Embodiment 4.
[1002] 8. The vector of Specific Embodiment 7 in which the
nucleotide sequence is multimerized.
[1003] 9. A host cell comprising the vector of Specific Embodiment
7.
[1004] 10. The host cell of Specific Embodiment 9 in which at least
one copy of said vector is integrated into the host cell
genome.
[1005] 11. The host cell of Specific Embodiment 10 in which two to
five copies of said vector are integrated into the host cell
genome.
[1006] 12. The host cell of Specific Embodiment 9 in which the
nucleotide sequence is multimerized.
[1007] 13. The host cell of Specific Embodiment 12 in which the
vector comprises two to five copies of said nucleotide
sequence.
[1008] 14. A cell genetically engineered to express the nucleic
acid molecule of Specific Embodiment 3.
[1009] 15. The cell of Specific Embodiment 14, which is a Pichia
pastoris cell.
[1010] 16. The cell of Specific Embodiment 15, wherein the
nucleotide sequence is operably linked to a methanol-inducible
promoter.
[1011] 17. A cell culture supernatant comprising the protein of
Specific Embodiment 1.
[1012] 18. A method of producing an elastase protein, comprising
culturing the cell of Specific Embodiment 15 under conditions in
which the protein of SEQ ID NO:64 or SEQ ID NO:69 is expressed.
[1013] 19. The method of Specific Embodiment 18, wherein said
conditions include one, two, three or all four of: (i) a period of
growth or induction at a pH of 2 to 6; (ii) a period of growth or
induction at a temperature of 22.degree. C. to 28.degree. C.; (iii)
culturing in complex medium; or (iv) culturing in the presence of a
citrate, succinate or acetate compound.
[1014] 20. The method of Specific Embodiment 18 which further
comprises recovering the protein.
[1015] 21. The method of Specific Embodiment 18, which further
comprises exposing said protein of SEQ ID NO:64 or SEQ ID NO:69 to
activation conditions to produce a mature elastase protein.
[1016] 22. The method of Specific Embodiment 21, wherein said
protein is purified prior to said exposure to activating
conditions.
[1017] 23. The method of Specific Embodiment 22, wherein said
protein is purified in the presence of a citrate, succinate or
acetate compound.
[1018] 24. The method of Specific Embodiment 21, further comprising
purifying the mature elastase protein.
[1019] 25. The method of Specific Embodiment 24, further comprising
lyophilizing the purified mature elastase protein.
[1020] 26. A method of making a mature elastase protein, comprising
subjecting a cell culture supernatant according to Specific
Embodiment 17 to autoactivation conditions, thereby producing a
mature elastase protein.
[1021] 27. The method of Specific Embodiment 26, further comprising
purifying the mature elastase protein.
[1022] 28. The method of Specific Embodiment 27, further comprising
lyophilizing the purified mature elastase protein.
[1023] 29. A method of making a pharmaceutical composition
comprising a mature elastase protein, reconstituting a lyophilisate
comprising the lyophilized proelastase protein produced by the
method of Specific Embodiment 25 or Specific Embodiment 28.
[1024] 30. The method of Specific Embodiment 29, wherein the
lyophilisate (a) comprises one or more buffer ingredients or (b)
does not comprise buffer ingredients.
[1025] 31. The method of Specific Embodiment 30, wherein the
lyophilisate is reconstituted with water or buffer.
[1026] 32. The method of Specific Embodiment 31, wherein upon
reconstitution a solution of mature elastase protein in full
strength buffer, greater than full strength buffer, or less than
full strength buffer is produced.
[1027] 33. The method of Specific Embodiment 32, wherein the buffer
is phosphate buffered saline.
[1028] 34. The method of Specific Embodiment 29, wherein the mature
elastase protein is reconstituted to a concentration of 0.001 mg/ml
to 50 mg/ml.
[1029] 35. The method of Specific Embodiment 29, wherein the mature
elastase protein has a specific activity of 1 to 40 U/mg
protein.
[1030] 36. A pharmaceutical composition produced by the method of
Specific Embodiment 29.
[1031] 37. The pharmaceutical composition of Specific Embodiment 36
which is characterized by at least one, at least two, at least
three, at least four, at least five, at least six or at least seven
of the following properties: [1032] (a) the composition is free of
trypsin; [1033] (b) the composition is substantially free of
trypsin; [1034] (c) the composition is free of any protein
consisting of SEQ ID NOS:70 and 71; [1035] (d) the composition is
substantially free of any protein consisting of SEQ ID NOS:2 and 3;
[1036] (e) the composition is free of bacterial proteins; [1037]
(f) the composition is substantially free of bacterial proteins;
[1038] (g) the composition is free of mammalian proteins other than
said mature elastase protein; [1039] (h) the composition is
substantially free of mammalian proteins other than said mature
elastase protein; [1040] (i) the composition is free or
substantially free of one, two, three or all four proteins
consisting of SEQ ID NO:85, 86, 94 and 95; [1041] (j) the
composition is free or substantially free of one, two, or all three
proteins consisting of SEQ ID NO:106, 107 and 108; [1042] (k) the
composition contains pharmaceutically acceptable levels of
endotoxins; [1043] (l) the mature elastase protein in the
composition is characterized by a specific activity of 1 to 40 U/mg
of protein; [1044] (m) the trypsin activity in said composition
corresponds to less than 4 ng per 1 mg of mature elastase protein;
[1045] (n) the composition comprises polysorbate-80; [1046] (o) the
composition comprises dextran; [1047] (p) the composition comprises
sodium ions, potassium ions, phosphate ions, chloride ions and
polysorbate-80; [1048] (q) the composition comprises sodium ions,
potassium ions, phosphate ions, chloride ions and dextran; [1049]
(r) the composition comprises sodium ions, potassium ions,
phosphate ions, chloride ions, polysorbate-80, and dextran; [1050]
(s) the mature elastase protein in said composition maintains 60%
to 100% of its specific activity after at least a week of storage
at 4.degree. C., after at least a month of storage at 4.degree. C.,
after at least two months of storage at 4.degree. C., after at
least three months of storage at 4.degree. C., or after at least
month six months of storage at 4.degree. C.; and [1051] (t) the
composition comprises a unit dosage of 0.0033 mg to 200 mg of said
mature elastase protein.
[1052] 38. The pharmaceutical composition of Specific Embodiment
37, wherein the pharmaceutical composition is characterized by at
least three characteristics, at least four characteristics or five
characteristics independently selected from the following groups
(i) through (v): [1053] (i) (a), (b) or (m) [1054] (ii) (e) or (f)
[1055] (iii) (g) or (h) [1056] (iv) (k) [1057] (v) (l)
[1058] 39. The pharmaceutical composition of 38, wherein two of
said at least three or at least said four characteristics are
selected from groups (i) and (iv) or (v).
[1059] 40. The pharmaceutical composition of 38, wherein three of
at least said four characteristics are selected from groups (i),
(iv) and (v).
[1060] 41. A method of removing one or more incorrectly processed
mature elastase proteins from a mixture of correctly and
incorrectly processed mature elastase proteins, said method
comprising: [1061] (a) subjecting a composition comprising a
mixture of correctly and incorrectly processed mature elastase
proteins to a pH at which the correctly processed mature enzyme is
active; [1062] (b) maintaining such pH until such time that one or
more incorrectly processed mature elastase proteins are
degraded,
[1063] thereby removing said one or more incorrectly processed
mature elastase proteins from a mixture of correctly and
incorrectly processed mature elastase proteins.
[1064] 42. The method of Specific Embodiment 41, wherein said one
or more incorrectly processed mature elastase proteins contain at
least one additional or fewer amino acid at the N-terminus relative
to correctly processed mature elastase proteins.
[1065] 43. The method of Specific Embodiment 41 wherein said pH is
between 5 and 12.
[1066] 44. The method of any one of Specific Embodiments 41,
wherein said one or more incorrectly processed mature elastase
proteins are degraded by 50% to 100%.
[1067] 45. A method for therapeutically increasing the diameter of
an artery or vein in a human subject in need thereof, the method
comprising: locally administering to the wall of the artery or vein
in the human subject the pharmaceutical composition of Specific
Embodiment 36 in a dose sufficient to increase the diameter of the
artery or vein.
[1068] 46. The method of Specific Embodiment 45, wherein the
diameter of the vessel, the lumenal diameter of the vessel, or
both, are increased.
[1069] 47. A method for preventing or treating vasospasm of an
artery or vein in a human subject in need thereof, the method
comprising: locally administering to the wall of the artery or vein
in the human subject the pharmaceutical composition of Specific
Embodiment 36 in a dose sufficient to prevent or treat vasospasm of
the artery or vein.
[1070] 48. A method for treating an obstructed artery or vein in a
human subject in need of such treatment, the method comprising:
locally administering to the wall of the artery or vein in the
human subject the pharmaceutical composition of Specific
Embodiments 36, wherein said administration results in proteolysis
of elastin in the wall of the artery or vein leading to enlargement
of the diameter of the artery or vein.
[1071] 49. A method for treating an artery or vein connected to an
arteriovenous hemodialysis graft or arteriovenous fistula in a
human subject in need of such treatment, the method comprising:
locally administering to the wall of the artery or vein in the
human subject the pharmaceutical composition of Specific Embodiment
36, wherein said administration results in proteolysis of elastin
in the wall of the artery or vein leading to enlargement of the
diameter of the artery or vein.
[1072] 50. A method for treating a vein in a human subject for use
in hemodialysis, the method comprising: locally administering to
the wall of the vein in the human subject the pharmaceutical
composition of Specific Embodiment 36, wherein said administration
results in proteolysis of elastin in the wall of the vein leading
to enlargement of the diameter of the vein.
[1073] 51. The kit comprising the pharmaceutical composition of
Specific Embodiment 36.
[1074] 52. The kit of Specific Embodiment 51 wherein the
pharmaceutical composition is in a container, pack, dispenser, or
catheter.
[1075] The claims of U.S. provisional application No. 60/992,319,
filed Dec. 4, 2007 are incorporated by reference herein in their
entireties, and each embodiment set forth in such claims is
incorporated by reference as a specific embodiment herein.
[1076] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims.
[1077] Various references, including patent applications, patents,
and scientific publications, are cited herein; the disclosure of
each such reference is hereby incorporated herein by reference in
its entirety.
Sequence CWU 1
1
1311240PRTHomo sapiensMOD_RES(202)..(202)Val or Leu 1Val Val Gly
Gly Thr Glu Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile1 5 10 15Ser Leu
Gln Tyr Arg Ser Gly Gly Ser Arg Tyr His Thr Cys Gly Gly 20 25 30Thr
Leu Ile Arg Gln Asn Trp Val Met Thr Ala Ala His Cys Val Asp 35 40
45Tyr Gln Lys Thr Phe Arg Val Val Ala Gly Asp His Asn Leu Ser Gln
50 55 60Asn Asp Gly Thr Glu Gln Tyr Val Ser Val Gln Lys Ile Val Val
His65 70 75 80Pro Tyr Trp Asn Ser Asp Asn Val Ala Ala Gly Tyr Asp
Ile Ala Leu 85 90 95Leu Arg Leu Ala Gln Ser Val Thr Leu Asn Ser Tyr
Val Gln Leu Gly 100 105 110Val Leu Pro Gln Glu Gly Ala Ile Leu Ala
Asn Asn Ser Pro Cys Tyr 115 120 125Ile Thr Gly Trp Gly Lys Thr Lys
Thr Asn Gly Gln Leu Ala Gln Thr 130 135 140Leu Gln Gln Ala Tyr Leu
Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser145 150 155 160Ser Ser Tyr
Trp Gly Ser Thr Val Lys Asn Thr Met Val Cys Ala Gly 165 170 175Gly
Asp Gly Val Arg Ser Gly Cys Gln Gly Asp Ser Gly Gly Pro Leu 180 185
190His Cys Leu Val Asn Gly Lys Tyr Ser Xaa His Gly Val Thr Ser Phe
195 200 205Val Ser Ser Arg Gly Cys Asn Val Ser Arg Lys Pro Thr Val
Phe Thr 210 215 220Gln Val Ser Ala Tyr Ile Ser Trp Ile Asn Asn Val
Ile Ala Ser Asn225 230 235 2402239PRTHomo
sapiensMOD_RES(201)..(201)Val or Leu 2Val Gly Gly Thr Glu Ala Gly
Arg Asn Ser Trp Pro Ser Gln Ile Ser1 5 10 15Leu Gln Tyr Arg Ser Gly
Gly Ser Arg Tyr His Thr Cys Gly Gly Thr 20 25 30Leu Ile Arg Gln Asn
Trp Val Met Thr Ala Ala His Cys Val Asp Tyr 35 40 45Gln Lys Thr Phe
Arg Val Val Ala Gly Asp His Asn Leu Ser Gln Asn 50 55 60Asp Gly Thr
Glu Gln Tyr Val Ser Val Gln Lys Ile Val Val His Pro65 70 75 80Tyr
Trp Asn Ser Asp Asn Val Ala Ala Gly Tyr Asp Ile Ala Leu Leu 85 90
95Arg Leu Ala Gln Ser Val Thr Leu Asn Ser Tyr Val Gln Leu Gly Val
100 105 110Leu Pro Gln Glu Gly Ala Ile Leu Ala Asn Asn Ser Pro Cys
Tyr Ile 115 120 125Thr Gly Trp Gly Lys Thr Lys Thr Asn Gly Gln Leu
Ala Gln Thr Leu 130 135 140Gln Gln Ala Tyr Leu Pro Ser Val Asp Tyr
Ala Ile Cys Ser Ser Ser145 150 155 160Ser Tyr Trp Gly Ser Thr Val
Lys Asn Thr Met Val Cys Ala Gly Gly 165 170 175Asp Gly Val Arg Ser
Gly Cys Gln Gly Asp Ser Gly Gly Pro Leu His 180 185 190Cys Leu Val
Asn Gly Lys Tyr Ser Xaa His Gly Val Thr Ser Phe Val 195 200 205Ser
Ser Arg Gly Cys Asn Val Ser Arg Lys Pro Thr Val Phe Thr Gln 210 215
220Val Ser Ala Tyr Ile Ser Trp Ile Asn Asn Val Ile Ala Ser Asn225
230 2353238PRTHomo sapiensMOD_RES(200)..(200)Val or Leu 3Gly Gly
Thr Glu Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile Ser Leu1 5 10 15Gln
Tyr Arg Ser Gly Gly Ser Arg Tyr His Thr Cys Gly Gly Thr Leu 20 25
30Ile Arg Gln Asn Trp Val Met Thr Ala Ala His Cys Val Asp Tyr Gln
35 40 45Lys Thr Phe Arg Val Val Ala Gly Asp His Asn Leu Ser Gln Asn
Asp 50 55 60Gly Thr Glu Gln Tyr Val Ser Val Gln Lys Ile Val Val His
Pro Tyr65 70 75 80Trp Asn Ser Asp Asn Val Ala Ala Gly Tyr Asp Ile
Ala Leu Leu Arg 85 90 95Leu Ala Gln Ser Val Thr Leu Asn Ser Tyr Val
Gln Leu Gly Val Leu 100 105 110Pro Gln Glu Gly Ala Ile Leu Ala Asn
Asn Ser Pro Cys Tyr Ile Thr 115 120 125Gly Trp Gly Lys Thr Lys Thr
Asn Gly Gln Leu Ala Gln Thr Leu Gln 130 135 140Gln Ala Tyr Leu Pro
Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser Ser145 150 155 160Tyr Trp
Gly Ser Thr Val Lys Asn Thr Met Val Cys Ala Gly Gly Asp 165 170
175Gly Val Arg Ser Gly Cys Gln Gly Asp Ser Gly Gly Pro Leu His Cys
180 185 190Leu Val Asn Gly Lys Tyr Ser Xaa His Gly Val Thr Ser Phe
Val Ser 195 200 205Ser Arg Gly Cys Asn Val Ser Arg Lys Pro Thr Val
Phe Thr Gln Val 210 215 220Ser Ala Tyr Ile Ser Trp Ile Asn Asn Val
Ile Ala Ser Asn225 230 2354240PRTHomo sapiensMOD_RES(202)..(202)Val
or Leu 4Ala Val Gly Gly Thr Glu Ala Gly Arg Asn Ser Trp Pro Ser Gln
Ile1 5 10 15Ser Leu Gln Tyr Arg Ser Gly Gly Ser Arg Tyr His Thr Cys
Gly Gly 20 25 30Thr Leu Ile Arg Gln Asn Trp Val Met Thr Ala Ala His
Cys Val Asp 35 40 45Tyr Gln Lys Thr Phe Arg Val Val Ala Gly Asp His
Asn Leu Ser Gln 50 55 60Asn Asp Gly Thr Glu Gln Tyr Val Ser Val Gln
Lys Ile Val Val His65 70 75 80Pro Tyr Trp Asn Ser Asp Asn Val Ala
Ala Gly Tyr Asp Ile Ala Leu 85 90 95Leu Arg Leu Ala Gln Ser Val Thr
Leu Asn Ser Tyr Val Gln Leu Gly 100 105 110Val Leu Pro Gln Glu Gly
Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr 115 120 125Ile Thr Gly Trp
Gly Lys Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr 130 135 140Leu Gln
Gln Ala Tyr Leu Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser145 150 155
160Ser Ser Tyr Trp Gly Ser Thr Val Lys Asn Thr Met Val Cys Ala Gly
165 170 175Gly Asp Gly Val Arg Ser Gly Cys Gln Gly Asp Ser Gly Gly
Pro Leu 180 185 190His Cys Leu Val Asn Gly Lys Tyr Ser Xaa His Gly
Val Thr Ser Phe 195 200 205Val Ser Ser Arg Gly Cys Asn Val Ser Arg
Lys Pro Thr Val Phe Thr 210 215 220Gln Val Ser Ala Tyr Ile Ser Trp
Ile Asn Asn Val Ile Ala Ser Asn225 230 235 2405186PRTHomo sapiens
5Val Val Gly Gly Thr Glu Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile1 5
10 15Ser Leu Gln Tyr Arg Ser Gly Gly Ser Arg Tyr His Thr Cys Gly
Gly 20 25 30Thr Leu Ile Arg Gln Asn Trp Val Met Thr Ala Ala His Cys
Val Asp 35 40 45Tyr Gln Lys Thr Phe Arg Val Val Ala Gly Asp His Asn
Leu Ser Gln 50 55 60Asn Asp Gly Thr Glu Gln Tyr Val Ser Val Gln Lys
Ile Val Val His65 70 75 80Pro Tyr Trp Asn Ser Asp Asn Val Ala Ala
Gly Tyr Asp Ile Ala Leu 85 90 95Leu Arg Leu Ala Gln Ser Val Thr Leu
Asn Ser Tyr Val Gln Leu Gly 100 105 110Val Leu Pro Gln Glu Gly Ala
Ile Leu Ala Asn Asn Ser Pro Cys Tyr 115 120 125Ile Thr Gly Trp Gly
Lys Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr 130 135 140Leu Gln Gln
Ala Tyr Leu Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser145 150 155
160Ser Ser Tyr Trp Gly Ser Thr Val Lys Asn Thr Met Val Cys Ala Gly
165 170 175Gly Asp Gly Ser Ser Leu Trp Met Pro Gly 180
1856250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 6Thr Gln Asp Leu Pro Glu Thr Asn Ala Ala Val
Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Arg Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Met Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln
Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala
Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr
Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Gln Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245 2507250PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
7Thr Gln Asp Leu Pro Glu Thr Asn Ala Ala Ala Val Gly Gly Thr Glu1 5
10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile Ser Leu Gln Tyr Arg
Ser 20 25 30Gly Gly Ser Arg Tyr His Thr Cys Gly Gly Thr Leu Ile Arg
Gln Asn 35 40 45Trp Val Met Thr Ala Ala His Cys Val Asp Tyr Gln Lys
Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn Leu Ser Gln Asn Asp
Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln Lys Ile Val Val His
Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala Gly Tyr Asp Ile Ala
Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr Leu Asn Ser Tyr Val
Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120 125Ala Ile Leu Ala Asn
Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys 130 135 140Thr Lys Thr
Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala Tyr Leu145 150 155
160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser Ser Tyr Trp Gly Ser
165 170 175Thr Val Lys Asn Thr Met Val Cys Ala Gly Gly Asp Gly Val
Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly Gly Pro Leu His Cys
Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His Gly Val Thr Ser Phe
Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser Arg Lys Pro Thr Val
Phe Thr Gln Val Ser Ala Tyr Ile225 230 235 240Ser Trp Ile Asn Asn
Val Ile Ala Ser Asn 245 2508250PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 8Thr Gln Asp Leu Pro Glu
Thr Ala Ala Ala Val Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser
Trp Pro Ser Gln Ile Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Arg
Tyr His Thr Cys Gly Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Met
Thr Ala Ala His Cys Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val
Ala Gly Asp His Asn Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75
80Tyr Val Ser Val Gln Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp
85 90 95Asn Val Ala Ala Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln
Ser 100 105 110Val Thr Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro
Gln Glu Gly 115 120 125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile
Thr Gly Trp Gly Lys 130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln
Thr Leu Gln Gln Ala Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala
Ile Cys Ser Ser Ser Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn
Thr Met Val Cys Ala Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys
Gln Gly Asp Ser Gly Gly Pro Leu His Cys Leu Val Asn Gly 195 200
205Lys Tyr Ser Xaa His Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys
210 215 220Asn Val Ser Arg Lys Pro Thr Val Phe Thr Gln Val Ser Ala
Tyr Ile225 230 235 240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
2509250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Thr Gln Asp Leu Pro Glu Thr Asn Asn Ala Pro
Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Arg Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Met Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln
Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala
Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr
Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Gln Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
25010250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 10Thr Gln Asp Leu Pro Glu Thr Asn Ala Arg Val
Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Arg Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Met Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln
Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala
Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr
Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Gln Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile
Ala Ser Asn 245 250113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 11Xaa Xaa
Xaa1123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Xaa Pro Xaa1133PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Xaa
Xaa Xaa1143PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Ala Ala Ala1153PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Asn
Ala Ala1163PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Asn Ala Pro1173PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Asn
Ala Arg1183PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Ala Pro Ala1193PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Ala
Ala Pro1203PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Asn Pro Ala1213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 21Leu
Pro Ala12210PRTHomo sapiens 22Thr Gln Asp Leu Pro Glu Thr Asn Ala
Arg1 5 102310PRTHomo sapiens 23Thr Gln Asp Leu Pro Glu Thr Asn Ala
Ala1 5 10248PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 24Glu Thr Asn Ala Arg Val Val Gly1
5258PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 25Thr Ala Ala Ala Val Val Gly Gly1
5268PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 26Thr Asn Ala Ala Ala Val Gly Gly1
5278PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Thr Asn Ala Ala Val Val Gly Gly1
5288PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Thr Asn Ala Pro Val Val Gly Gly1
5298PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 29Thr Gly Ala Gly Ile Val Gly Gly1
5308PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Thr Val Pro Gly Val Val Gly Gly1
5318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Thr Ala Pro Gly Val Val Gly Gly1
5328PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 32Thr Asn Pro Gly Val Val Gly Gly1 533753DNAHomo
sapiens 33acccaggacc ttccggaaac caatgcccgc gtagtcggag ggactgaggc
cgggaggaat 60tcctggccct ctcagatttc cctccagtac cggtctggag gttcccggta
tcacacctgt 120ggagggaccc ttatcagaca gaactgggtg atgacagctg
ctcactgcgt ggattaccag 180aagactttcc gcgtggtggc tggagaccat
aacctgagcc agaatgatgg cactgagcag 240tacgtgagtg tgcagaagat
cgtggtgcat ccatactgga acagcgataa cgtggctgcc 300ggctatgaca
tcgccctgct gcgcctggcc cagagcgtta ccctcaatag ctatgtccag
360ctgggtgttc tgccccagga gggagccatc ctggctaaca acagtccctg
ctacatcaca 420ggctggggca agaccaagac caatgggcag ctggcccaga
ccctgcagca ggcttacctg 480ccctctgtgg actacgccat ctgctccagc
tcctcctact ggggctccac tgtgaagaac 540accatggtgt gtgctggtgg
agatggagtt cgctctggat gccagggtga ctctgggggc 600cccctccatt
gcttggtgaa tggcaagtat tctgtccatg gagtgaccag ctttgtgtcc
660agccggggct gtaatgtctc caggaagcct acagtcttca cccaggtctc
tgcttacatc 720tcctggataa ataatgtcat cgcctccaac tga
7533424PRTUnknownDescription of Unknown Yeast alpha factor signal
peptide 34Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala
Ser Ser1 5 10 15Ala Leu Ala Ala Pro Val Asn Thr 203556DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
35ggctcgagaa aagagaggct gaagctactc aggaccttcc ggaaaccaat gcccgg
563633DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 36gggccgcggc ttatcagttg gaggcgatga cat
3337242PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 37Ala Ala Val Val Gly Gly Thr Glu Ala Gly Arg
Asn Ser Trp Pro Ser1 5 10 15Gln Ile Ser Leu Gln Tyr Arg Ser Gly Gly
Ser Arg Tyr His Thr Cys 20 25 30Gly Gly Thr Leu Ile Arg Gln Asn Trp
Val Met Thr Ala Ala His Cys 35 40 45Val Asp Tyr Gln Lys Thr Phe Arg
Val Val Ala Gly Asp His Asn Leu 50 55 60Ser Gln Asn Asp Gly Thr Glu
Gln Tyr Val Ser Val Gln Lys Ile Val65 70 75 80Val His Pro Tyr Trp
Asn Ser Asp Asn Val Ala Ala Gly Tyr Asp Ile 85 90 95Ala Leu Leu Arg
Leu Ala Gln Ser Val Thr Leu Asn Ser Tyr Val Gln 100 105 110Leu Gly
Val Leu Pro Gln Glu Gly Ala Ile Leu Ala Asn Asn Ser Pro 115 120
125Cys Tyr Ile Thr Gly Trp Gly Lys Thr Lys Thr Asn Gly Gln Leu Ala
130 135 140Gln Thr Leu Gln Gln Ala Tyr Leu Pro Ser Val Asp Tyr Ala
Ile Cys145 150 155 160Ser Ser Ser Ser Tyr Trp Gly Ser Thr Val Lys
Asn Thr Met Val Cys 165 170 175Ala Gly Gly Asp Gly Val Arg Ser Gly
Cys Gln Gly Asp Ser Gly Gly 180 185 190Pro Leu His Cys Leu Val Asn
Gly Lys Tyr Ser Xaa His Gly Val Thr 195 200 205Ser Phe Val Ser Ser
Arg Gly Cys Asn Val Ser Arg Lys Pro Thr Val 210 215 220Phe Thr Gln
Val Ser Ala Tyr Ile Ser Trp Ile Asn Asn Val Ile Ala225 230 235
240Ser Asn38241PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 38Ala Val Val Gly Gly Thr Glu Ala
Gly Arg Asn Ser Trp Pro Ser Gln1 5 10 15Ile Ser Leu Gln Tyr Arg Ser
Gly Gly Ser Arg Tyr His Thr Cys Gly 20 25 30Gly Thr Leu Ile Arg Gln
Asn Trp Val Met Thr Ala Ala His Cys Val 35 40 45Asp Tyr Gln Lys Thr
Phe Arg Val Val Ala Gly Asp His Asn Leu Ser 50 55 60Gln Asn Asp Gly
Thr Glu Gln Tyr Val Ser Val Gln Lys Ile Val Val65 70 75 80His Pro
Tyr Trp Asn Ser Asp Asn Val Ala Ala Gly Tyr Asp Ile Ala 85 90 95Leu
Leu Arg Leu Ala Gln Ser Val Thr Leu Asn Ser Tyr Val Gln Leu 100 105
110Gly Val Leu Pro Gln Glu Gly Ala Ile Leu Ala Asn Asn Ser Pro Cys
115 120 125Tyr Ile Thr Gly Trp Gly Lys Thr Lys Thr Asn Gly Gln Leu
Ala Gln 130 135 140Thr Leu Gln Gln Ala Tyr Leu Pro Ser Val Asp Tyr
Ala Ile Cys Ser145 150 155 160Ser Ser Ser Tyr Trp Gly Ser Thr Val
Lys Asn Thr Met Val Cys Ala 165 170 175Gly Gly Asp Gly Val Arg Ser
Gly Cys Gln Gly Asp Ser Gly Gly Pro 180 185 190Leu His Cys Leu Val
Asn Gly Lys Tyr Ser Xaa His Gly Val Thr Ser 195 200 205Phe Val Ser
Ser Arg Gly Cys Asn Val Ser Arg Lys Pro Thr Val Phe 210 215 220Thr
Gln Val Ser Ala Tyr Ile Ser Trp Ile Asn Asn Val Ile Ala Ser225 230
235 240Asn39240PRTSus scrofa 39Val Val Gly Gly Thr Glu Ala Gln Arg
Asn Ser Trp Pro Ser Gln Ile1 5 10 15Ser Leu Gln Tyr Arg Ser Gly Ser
Ser Trp Ala His Thr Cys Gly Gly 20 25 30Thr Leu Ile Arg Gln Asn Trp
Val Met Thr Ala Ala His Cys Val Asp 35 40 45Arg Glu Leu Thr Phe Arg
Val Val Val Gly Glu His Asn Leu Asn Gln 50 55 60Asn Asp Gly Thr Glu
Gln Tyr Val Gly Val Gln Lys Ile Val Val His65 70 75 80Pro Tyr Trp
Asn Thr Asp Asp Val Ala Ala Gly Tyr Asp Ile Ala Leu 85 90 95Leu Arg
Leu Ala Gln Ser Val Thr Leu Asn Ser Tyr Val Gln Leu Gly 100 105
110Val Leu Pro Arg Ala Gly Thr Ile Leu Ala Asn Asn Ser Pro Cys Tyr
115 120 125Ile Thr Gly Trp Gly Leu Thr Arg Thr Asn Gly Gln Leu Ala
Gln Thr 130 135 140Leu Gln Gln Ala Tyr Leu Pro Thr Val Asp Tyr Ala
Ile Cys Ser Ser145 150 155 160Ser Ser Tyr Trp Gly Ser Thr Val Lys
Asn Ser Met Val Cys Ala Gly 165 170 175Gly Asp Gly Val Arg Ser Gly
Cys Gln Gly Asp Ser Gly Gly Pro Leu 180 185 190His Cys Leu Val Asn
Gly Gln Tyr Ala Val His Gly Val Thr Ser Phe 195 200 205Val Ser Arg
Leu Gly Cys Asn Val Thr Arg Lys Pro Thr Val Phe Thr 210 215 220Arg
Val Ser Ala Tyr Ile Ser Trp Ile Asn Asn Val Ile Ala Ser Asn225 230
235 240408PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 40Glu Thr Ala Ala Ala Val Val Gly1
5418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 41Glu Thr Asn Ala Ala Ala Val Gly1
5428PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42Glu Thr Asn Ala Ala Val Val Gly1
5438PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 43Glu Thr Asn Ala Pro Val Val Gly1
5448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Glu Thr Gly Ala Gly Ile Val Gly1
5458PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45Glu Thr Val Pro Gly Val Val Gly1
5468PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Glu Thr Ala Pro Gly Val Val Gly1
5478PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 47Glu Thr Asn Pro Gly Val Val Gly1
5488PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Glu Thr Asn Pro Ala Val Val Gly1
5498PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 49Glu Thr Asn His Ala Val Val Gly1
55090PRTUnknownDescription of Unknown Yeast alpha-mating factor
signal peptide, propeptide, and spacer sequence 1 50Met Arg Phe Pro
Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala
Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30Ile Pro
Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe 35 40 45Asp
Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Asn Gly Leu 50 55
60Leu Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly65
70 75 80Val Ser Leu Asp Lys Arg Glu Ala Glu Ala 85
905184PRTUnknownDescription of Unknown Yeast alpha-mating factor
signal peptide and propeptide sequence 2 51Met Arg Phe Pro Ser Ile
Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Ala Pro
Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30Ile Pro Ala Glu
Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe 35 40 45Asp Val Ala
Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Asn Gly Leu 50 55 60Leu Phe
Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly65 70 75
80Val Ser Leu Asp528PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 52Glu Thr Lys Pro Ala Val Val Gly1
5538PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Glu Thr His Pro Ala Val Val Gly1
5548PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 54Glu His Asn Pro Ala Val Val Gly1
5558PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 55His Thr Asn Pro Ala Val Val Gly1
5568PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Pro Thr His Pro Ala Val Val Gly1
5578PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 57Pro Thr Asn Pro Ala Val Val Gly1
5588PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 58His Thr His Pro Ala Val Val Gly1
5598PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 59Glu Thr Phe Pro Ala Val Val Gly1
5608PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60His Thr Phe Pro Ala Val Val Gly1
5618PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 61Gly Thr Phe Pro Ala Val Val Gly1
5628PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 62His Thr Gly Pro Ala Val Val Gly1
5638PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 63His Thr Lys Pro Ala Val Val Gly1
564250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 64Thr Gln Asp Leu Pro Glu Thr Asn Pro Ala Val
Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Arg Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Met Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln
Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala
Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr
Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Gln Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
25065250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 65Thr Gln Asp Leu Pro Glu Thr Asn His Ala Val
Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Arg Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Met Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln
Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala
Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr
Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Gln Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
25066250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 66Thr Gln Asp Leu Pro Glu Thr Lys Pro Ala Val
Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Arg Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Met Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln
Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala
Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr
Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Gln Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
25067250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 67Thr Gln Asp Leu Pro Glu Thr His Pro Ala Val
Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Arg Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Met Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln
Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala
Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr
Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Gln Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
25068250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 68Thr Gln Asp Leu Pro Glu His Asn Pro Ala Val
Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Arg Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Met Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln
Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala
Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr
Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Gln Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
25069250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 69Thr Gln Asp Leu Pro His Thr Asn Pro Ala Val
Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Arg Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Met Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln
Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala
Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr
Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Gln Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245 25070242PRTHomo
sapiensMOD_RES(204)..(204)Val or Leu 70Ala Arg Val Val Gly Gly Thr
Glu Ala Gly Arg Asn Ser Trp Pro Ser1 5 10 15Gln Ile Ser Leu Gln Tyr
Arg Ser Gly Gly Ser Arg Tyr His Thr Cys 20 25 30Gly Gly Thr Leu Ile
Arg Gln Asn Trp Val Met Thr Ala Ala His Cys 35 40 45Val Asp Tyr Gln
Lys Thr Phe Arg Val Val Ala Gly Asp His Asn Leu 50 55 60Ser Gln Asn
Asp Gly Thr Glu Gln Tyr Val Ser Val Gln Lys Ile Val65 70 75 80Val
His Pro Tyr Trp Asn Ser Asp Asn Val Ala Ala Gly Tyr Asp Ile 85 90
95Ala Leu Leu Arg Leu Ala Gln Ser Val Thr Leu Asn Ser Tyr Val Gln
100 105 110Leu Gly Val Leu Pro Gln Glu Gly Ala Ile Leu Ala Asn Asn
Ser Pro 115 120 125Cys Tyr Ile Thr Gly Trp Gly Lys Thr Lys Thr Asn
Gly Gln Leu Ala 130 135 140Gln Thr Leu Gln Gln Ala Tyr Leu Pro Ser
Val Asp Tyr Ala Ile Cys145 150 155 160Ser Ser Ser Ser Tyr Trp Gly
Ser Thr Val Lys Asn Thr Met Val Cys 165 170 175Ala Gly Gly Asp Gly
Val Arg Ser Gly Cys Gln Gly Asp Ser Gly Gly 180 185 190Pro Leu His
Cys Leu Val Asn Gly Lys Tyr Ser Xaa His Gly Val Thr 195 200 205Ser
Phe Val Ser Ser Arg Gly Cys Asn Val Ser Arg Lys Pro Thr Val 210 215
220Phe Thr Gln Val Ser Ala Tyr Ile Ser Trp Ile Asn Asn Val Ile
Ala225 230 235 240Ser Asn71241PRTHomo sapiensMOD_RES(203)..(203)Val
or Leu 71Arg Val Val Gly Gly Thr Glu Ala Gly Arg Asn Ser Trp Pro
Ser Gln1 5 10 15Ile Ser Leu Gln Tyr Arg Ser Gly Gly Ser Arg Tyr His
Thr Cys Gly 20 25 30Gly Thr Leu Ile Arg Gln Asn Trp Val Met Thr Ala
Ala His Cys Val 35 40 45Asp Tyr Gln Lys Thr Phe Arg Val Val Ala Gly
Asp His Asn Leu Ser 50 55 60Gln Asn Asp Gly Thr Glu Gln Tyr Val Ser
Val Gln Lys Ile Val Val65 70 75 80His Pro Tyr Trp Asn Ser Asp Asn
Val Ala Ala Gly Tyr Asp Ile Ala 85 90 95Leu Leu Arg Leu Ala Gln Ser
Val Thr Leu Asn Ser Tyr Val Gln Leu 100 105 110Gly Val Leu Pro Gln
Glu Gly Ala Ile Leu Ala Asn Asn Ser Pro Cys 115 120 125Tyr Ile Thr
Gly Trp Gly Lys Thr Lys Thr Asn Gly Gln Leu Ala Gln 130 135 140Thr
Leu Gln Gln Ala Tyr Leu Pro Ser Val Asp Tyr Ala Ile Cys Ser145 150
155 160Ser Ser Ser Tyr Trp Gly Ser Thr Val Lys Asn Thr Met Val Cys
Ala 165 170 175Gly Gly Asp Gly Val Arg Ser Gly Cys Gln Gly Asp Ser
Gly Gly Pro 180 185 190Leu His Cys Leu Val Asn Gly Lys Tyr Ser Xaa
His Gly Val Thr Ser 195 200 205Phe Val Ser Ser Arg Gly Cys Asn Val
Ser Arg Lys Pro Thr Val Phe 210 215 220Thr Gln Val Ser Ala Tyr Ile
Ser Trp Ile Asn Asn Val Ile Ala Ser225 230 235 240Asn7210PRTHomo
sapiens 72Thr Gln Asp Leu Pro Glu Thr Asn Pro Ala1 5 107310PRTHomo
sapiens 73Thr Gln Asp Leu Pro His Thr Asn Pro Ala1 5 10748PRTHomo
sapiensMOD_RES(1)..(1)Glu, His, Pro, Gly, Asp, Lys, or Ala 74Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 57527DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 75atctacgtag tcggagggac
tgaggcc 277628DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 76gtcgacaagc ttatcagttg gaggcgat
2877214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 77Val Val Gly Gly Thr Glu Ala Gly Arg Asn Ser
Trp Pro Ser Gln Ile1 5 10 15Ser Leu Gln Tyr Arg Ser Gly Gly Ser Arg
Tyr His Thr Cys Gly Gly 20 25 30Thr Leu Ile Arg Gln Asn Trp Val Met
Thr Ala Ala His Cys Val Asp 35 40 45Tyr Gln Lys Thr Phe Arg Val Val
Ala Gly Asp His Asn Leu Ser Gln 50 55 60Asn Asp Gly Thr Glu Gln Tyr
Val Ser Val Gln Lys Ile Val Val His65 70 75 80Pro Tyr Trp Asn Ser
Asp Asn Val Ala Ala Gly Tyr Asp Ile Ala Leu 85 90 95Leu Arg Leu Ala
Gln Ser Val Thr Leu Asn Ser Tyr Val Gln Leu Gly 100 105 110Val Leu
Pro Gln Glu Gly Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr 115 120
125Ile Thr Gly Trp Gly Lys Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr
130 135 140Leu Gln Gln Ala Tyr Leu Pro Ser Val Asp Tyr Ala Ile Cys
Ser Ser145 150 155 160Ser Ser Tyr Trp Gly Ser Thr Val Lys Asn Thr
Met Val Cys Ala Gly 165 170 175Gly Asp Gly Val Arg Ser Gly Cys Gln
Gly Asp Ser Gly Gly Pro Pro 180 185 190Pro Leu Leu Gly Glu Trp Gln
Val Phe Ser Pro Trp Ser Asp Gln Leu 195 200 205Cys Val Gln Pro Gly
Leu 21078240PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 78Val Val Gly Gly Thr Glu Ala Gly
Arg Asn Ser Trp Pro Ser Gln Ile1 5 10 15Ser Leu Gln Tyr Arg Ser Gly
Gly Ser Xaa Tyr His Thr Cys Gly Gly 20 25 30Thr Leu Ile Arg Gln Asn
Trp Val Xaa Thr Ala Ala His Cys Val Asp 35 40 45Tyr Gln Lys Thr Phe
Arg Val Val Ala Gly Asp His Asn Leu Ser Gln 50 55 60Asn Asp Gly Thr
Glu Gln Tyr Val Ser Val Gln Lys Ile Val Val His65 70 75 80Pro Tyr
Trp Asn Ser Asp Asn Val Ala Ala Gly Tyr Asp Ile Ala Leu 85 90 95Leu
Arg Leu Ala Gln Ser Val Thr Leu Asn Ser Tyr Val Gln Leu Gly 100 105
110Val Leu Pro Gln Glu Gly Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr
115 120 125Ile Thr Gly Trp Gly Lys Thr Lys Thr Asn Gly Gln Leu Ala
Gln Thr 130 135 140Leu Gln Gln Ala Tyr Leu Pro Ser Val Asp Tyr Ala
Ile Cys Ser Ser145 150 155 160Ser Ser Tyr Trp Gly Ser Thr Val Lys
Asn Thr Met Val Cys Ala Gly 165 170 175Gly Asp Gly Val Arg Ser Gly
Cys Gln Gly Asp Ser Gly Gly Pro Leu 180 185 190His Cys Leu Val Asn
Gly Lys Tyr Ser Xaa His Gly Val Thr Ser Phe 195 200 205Val Ser Ser
Arg Gly Cys Asn Val Ser Arg Lys Pro Thr Val Phe Thr 210 215 220Xaa
Val Ser Ala Tyr Ile Ser Trp Ile Asn Asn Val Ile Ala Ser Asn225 230
235 2407910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 79Thr Xaa Asp Leu Pro Glu Thr Asn Ala Arg1 5
108010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 80Thr Xaa Asp Leu Pro Xaa Xaa Xaa Xaa Xaa1 5
1081756DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 81actcaggacc ttccggaaac caatgcccgg
gtagtcggag ggactgaggc cgggaggaac 60tcctggccct ctcagatttc cctccagtac
cggtctggag gttcctggta tcacacctgt 120ggagggaccc ttatcagaca
gaactgggtg atgacagctg cacactgcgt ggattaccag 180aagactttcc
gcgtggtggc tggagaccat aacctgagcc agaatgatgg cactgagcag
240tacgtgagtg tgcagaagat cgtggtgcat ccatactgga acagcgataa
cgtggctgca 300ggctatgaca tcgccctgct gcgcctggcc cagagcgtta
ccctcaatag ctatgtccag 360ctgggtgttc tgccccagga gggagccatc
ctggctaaca acagtccctg ctacatcaca 420ggctggggca agaccaagac
caatgggcag ctggcccaga ccttgcagca ggcttacctg 480ccctctgtgg
actatgccat ctgctccagc tcctcctact ggggctccac tgtgaagaac
540actatggtgt gtgctggtgg agatggagtt cgctctggat gtcagggtga
ctctgggggc 600cccctccatt gcttggtgaa tggcaagtat tctcttcatg
gagtgaccag ctttgtgtcc 660agccggggct gtaatgtctc tagaaagcct
acagtcttca cacgggtctc tgcttacatc 720tcctggataa ataatgtcat
cgcctccaac tgataa 75682250PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 82Thr Gln Asp Leu Pro Glu
Thr Asn Ala Arg Val Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser
Trp Pro Ser Gln Ile Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Trp
Tyr His Thr Cys Gly Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Met
Thr Ala Ala His Cys Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val
Ala Gly Asp His Asn Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75
80Tyr Val Ser Val Gln Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp
85 90 95Asn Val Ala Ala Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln
Ser 100 105 110Val Thr Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro
Gln Glu Gly 115 120 125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile
Thr Gly Trp Gly Lys 130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln
Thr Leu Gln Gln Ala Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala
Ile Cys Ser Ser
Ser Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys
Ala Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser
Gly Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Leu
His Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val
Ser Arg Lys Pro Thr Val Phe Thr Arg Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
25083250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 83Thr Xaa Asp Leu Pro Glu Thr Asn Ala Arg Val
Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Xaa Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Xaa Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln
Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala
Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr
Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Xaa Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245 25084240PRTHomo
sapiensMOD_RES(26)..(26)Trp or Arg 84Val Val Gly Gly Thr Glu Ala
Gly Arg Asn Ser Trp Pro Ser Gln Ile1 5 10 15Ser Leu Gln Tyr Arg Ser
Gly Gly Ser Xaa Tyr His Thr Cys Gly Gly 20 25 30Thr Leu Ile Arg Gln
Asn Trp Val Xaa Thr Ala Ala His Cys Val Asp 35 40 45Tyr Gln Lys Thr
Phe Arg Val Val Ala Gly Asp His Asn Leu Ser Gln 50 55 60Asn Asp Gly
Thr Glu Gln Tyr Val Ser Val Gln Lys Ile Val Val His65 70 75 80Pro
Tyr Trp Asn Ser Asp Asn Val Ala Ala Gly Tyr Asp Ile Ala Leu 85 90
95Leu Arg Leu Ala Gln Ser Val Thr Leu Asn Ser Tyr Val Gln Leu Gly
100 105 110Val Leu Pro Gln Glu Gly Ala Ile Leu Ala Asn Asn Ser Pro
Cys Tyr 115 120 125Ile Thr Gly Trp Gly Lys Thr Lys Thr Asn Gly Gln
Leu Ala Gln Thr 130 135 140Leu Gln Gln Ala Tyr Leu Pro Ser Val Asp
Tyr Ala Ile Cys Ser Ser145 150 155 160Ser Ser Tyr Trp Gly Ser Thr
Val Lys Asn Thr Met Val Cys Ala Gly 165 170 175Gly Asp Gly Val Arg
Ser Gly Cys Gln Gly Asp Ser Gly Gly Pro Leu 180 185 190His Cys Leu
Val Asn Gly Lys Tyr Ser Xaa His Gly Val Thr Ser Phe 195 200 205Val
Ser Ser Arg Gly Cys Asn Val Ser Arg Lys Pro Thr Val Phe Thr 210 215
220Xaa Val Ser Ala Tyr Ile Ser Trp Ile Asn Asn Val Ile Ala Ser
Asn225 230 235 24085239PRTHomo sapiensMOD_RES(25)..(25)Trp or Arg
85Val Gly Gly Thr Glu Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile Ser1
5 10 15Leu Gln Tyr Arg Ser Gly Gly Ser Xaa Tyr His Thr Cys Gly Gly
Thr 20 25 30Leu Ile Arg Gln Asn Trp Val Xaa Thr Ala Ala His Cys Val
Asp Tyr 35 40 45Gln Lys Thr Phe Arg Val Val Ala Gly Asp His Asn Leu
Ser Gln Asn 50 55 60Asp Gly Thr Glu Gln Tyr Val Ser Val Gln Lys Ile
Val Val His Pro65 70 75 80Tyr Trp Asn Ser Asp Asn Val Ala Ala Gly
Tyr Asp Ile Ala Leu Leu 85 90 95Arg Leu Ala Gln Ser Val Thr Leu Asn
Ser Tyr Val Gln Leu Gly Val 100 105 110Leu Pro Gln Glu Gly Ala Ile
Leu Ala Asn Asn Ser Pro Cys Tyr Ile 115 120 125Thr Gly Trp Gly Lys
Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu 130 135 140Gln Gln Ala
Tyr Leu Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser145 150 155
160Ser Tyr Trp Gly Ser Thr Val Lys Asn Thr Met Val Cys Ala Gly Gly
165 170 175Asp Gly Val Arg Ser Gly Cys Gln Gly Asp Ser Gly Gly Pro
Leu His 180 185 190Cys Leu Val Asn Gly Lys Tyr Ser Xaa His Gly Val
Thr Ser Phe Val 195 200 205Ser Ser Arg Gly Cys Asn Val Ser Arg Lys
Pro Thr Val Phe Thr Xaa 210 215 220Val Ser Ala Tyr Ile Ser Trp Ile
Asn Asn Val Ile Ala Ser Asn225 230 23586238PRTHomo
sapiensMOD_RES(24)..(24)Trp or Arg 86Gly Gly Thr Glu Ala Gly Arg
Asn Ser Trp Pro Ser Gln Ile Ser Leu1 5 10 15Gln Tyr Arg Ser Gly Gly
Ser Xaa Tyr His Thr Cys Gly Gly Thr Leu 20 25 30Ile Arg Gln Asn Trp
Val Xaa Thr Ala Ala His Cys Val Asp Tyr Gln 35 40 45Lys Thr Phe Arg
Val Val Ala Gly Asp His Asn Leu Ser Gln Asn Asp 50 55 60Gly Thr Glu
Gln Tyr Val Ser Val Gln Lys Ile Val Val His Pro Tyr65 70 75 80Trp
Asn Ser Asp Asn Val Ala Ala Gly Tyr Asp Ile Ala Leu Leu Arg 85 90
95Leu Ala Gln Ser Val Thr Leu Asn Ser Tyr Val Gln Leu Gly Val Leu
100 105 110Pro Gln Glu Gly Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr
Ile Thr 115 120 125Gly Trp Gly Lys Thr Lys Thr Asn Gly Gln Leu Ala
Gln Thr Leu Gln 130 135 140Gln Ala Tyr Leu Pro Ser Val Asp Tyr Ala
Ile Cys Ser Ser Ser Ser145 150 155 160Tyr Trp Gly Ser Thr Val Lys
Asn Thr Met Val Cys Ala Gly Gly Asp 165 170 175Gly Val Arg Ser Gly
Cys Gln Gly Asp Ser Gly Gly Pro Leu His Cys 180 185 190Leu Val Asn
Gly Lys Tyr Ser Xaa His Gly Val Thr Ser Phe Val Ser 195 200 205Ser
Arg Gly Cys Asn Val Ser Arg Lys Pro Thr Val Phe Thr Xaa Val 210 215
220Ser Ala Tyr Ile Ser Trp Ile Asn Asn Val Ile Ala Ser Asn225 230
23587240PRTHomo sapiensMOD_RES(26)..(26)Trp or Arg 87Ala Val Gly
Gly Thr Glu Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile1 5 10 15Ser Leu
Gln Tyr Arg Ser Gly Gly Ser Xaa Tyr His Thr Cys Gly Gly 20 25 30Thr
Leu Ile Arg Gln Asn Trp Val Xaa Thr Ala Ala His Cys Val Asp 35 40
45Tyr Gln Lys Thr Phe Arg Val Val Ala Gly Asp His Asn Leu Ser Gln
50 55 60Asn Asp Gly Thr Glu Gln Tyr Val Ser Val Gln Lys Ile Val Val
His65 70 75 80Pro Tyr Trp Asn Ser Asp Asn Val Ala Ala Gly Tyr Asp
Ile Ala Leu 85 90 95Leu Arg Leu Ala Gln Ser Val Thr Leu Asn Ser Tyr
Val Gln Leu Gly 100 105 110Val Leu Pro Gln Glu Gly Ala Ile Leu Ala
Asn Asn Ser Pro Cys Tyr 115 120 125Ile Thr Gly Trp Gly Lys Thr Lys
Thr Asn Gly Gln Leu Ala Gln Thr 130 135 140Leu Gln Gln Ala Tyr Leu
Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser145 150 155 160Ser Ser Tyr
Trp Gly Ser Thr Val Lys Asn Thr Met Val Cys Ala Gly 165 170 175Gly
Asp Gly Val Arg Ser Gly Cys Gln Gly Asp Ser Gly Gly Pro Leu 180 185
190His Cys Leu Val Asn Gly Lys Tyr Ser Xaa His Gly Val Thr Ser Phe
195 200 205Val Ser Ser Arg Gly Cys Asn Val Ser Arg Lys Pro Thr Val
Phe Thr 210 215 220Xaa Val Ser Ala Tyr Ile Ser Trp Ile Asn Asn Val
Ile Ala Ser Asn225 230 235 24088250PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
88Thr Xaa Asp Leu Pro Glu Thr Asn Ala Ala Val Val Gly Gly Thr Glu1
5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile Ser Leu Gln Tyr Arg
Ser 20 25 30Gly Gly Ser Xaa Tyr His Thr Cys Gly Gly Thr Leu Ile Arg
Gln Asn 35 40 45Trp Val Xaa Thr Ala Ala His Cys Val Asp Tyr Gln Lys
Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn Leu Ser Gln Asn Asp
Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln Lys Ile Val Val His
Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala Gly Tyr Asp Ile Ala
Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr Leu Asn Ser Tyr Val
Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120 125Ala Ile Leu Ala Asn
Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys 130 135 140Thr Lys Thr
Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala Tyr Leu145 150 155
160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser Ser Tyr Trp Gly Ser
165 170 175Thr Val Lys Asn Thr Met Val Cys Ala Gly Gly Asp Gly Val
Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly Gly Pro Leu His Cys
Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His Gly Val Thr Ser Phe
Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser Arg Lys Pro Thr Val
Phe Thr Xaa Val Ser Ala Tyr Ile225 230 235 240Ser Trp Ile Asn Asn
Val Ile Ala Ser Asn 245 25089250PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 89Thr Xaa Asp Leu Pro
Glu Thr Asn Ala Ala Ala Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn
Ser Trp Pro Ser Gln Ile Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser
Xaa Tyr His Thr Cys Gly Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val
Xaa Thr Ala Ala His Cys Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val
Val Ala Gly Asp His Asn Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75
80Tyr Val Ser Val Gln Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp
85 90 95Asn Val Ala Ala Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln
Ser 100 105 110Val Thr Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro
Gln Glu Gly 115 120 125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile
Thr Gly Trp Gly Lys 130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln
Thr Leu Gln Gln Ala Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala
Ile Cys Ser Ser Ser Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn
Thr Met Val Cys Ala Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys
Gln Gly Asp Ser Gly Gly Pro Leu His Cys Leu Val Asn Gly 195 200
205Lys Tyr Ser Xaa His Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys
210 215 220Asn Val Ser Arg Lys Pro Thr Val Phe Thr Xaa Val Ser Ala
Tyr Ile225 230 235 240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
25090250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 90Thr Xaa Asp Leu Pro Glu Thr Ala Ala Ala Val
Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Xaa Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Xaa Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln
Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala
Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr
Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Xaa Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
25091250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 91Thr Xaa Asp Leu Pro Glu Thr Asn Asn Ala Pro
Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Xaa Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Xaa Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln
Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala
Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr
Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Xaa Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
25092250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 92Thr Xaa Asp Leu Pro Glu Thr Asn Ala Arg Val
Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Xaa Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Xaa Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln
Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp
85 90 95Asn Val Ala Ala Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln
Ser 100 105 110Val Thr Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro
Gln Glu Gly 115 120 125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile
Thr Gly Trp Gly Lys 130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln
Thr Leu Gln Gln Ala Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala
Ile Cys Ser Ser Ser Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn
Thr Met Val Cys Ala Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys
Gln Gly Asp Ser Gly Gly Pro Leu His Cys Leu Val Asn Gly 195 200
205Lys Tyr Ser Xaa His Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys
210 215 220Asn Val Ser Arg Lys Pro Thr Val Phe Thr Xaa Val Ser Ala
Tyr Ile225 230 235 240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
250933PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 93Xaa Pro Xaa194242PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
94Ala Ala Val Val Gly Gly Thr Glu Ala Gly Arg Asn Ser Trp Pro Ser1
5 10 15Gln Ile Ser Leu Gln Tyr Arg Ser Gly Gly Ser Xaa Tyr His Thr
Cys 20 25 30Gly Gly Thr Leu Ile Arg Gln Asn Trp Val Xaa Thr Ala Ala
His Cys 35 40 45Val Asp Tyr Gln Lys Thr Phe Arg Val Val Ala Gly Asp
His Asn Leu 50 55 60Ser Gln Asn Asp Gly Thr Glu Gln Tyr Val Ser Val
Gln Lys Ile Val65 70 75 80Val His Pro Tyr Trp Asn Ser Asp Asn Val
Ala Ala Gly Tyr Asp Ile 85 90 95Ala Leu Leu Arg Leu Ala Gln Ser Val
Thr Leu Asn Ser Tyr Val Gln 100 105 110Leu Gly Val Leu Pro Gln Glu
Gly Ala Ile Leu Ala Asn Asn Ser Pro 115 120 125Cys Tyr Ile Thr Gly
Trp Gly Lys Thr Lys Thr Asn Gly Gln Leu Ala 130 135 140Gln Thr Leu
Gln Gln Ala Tyr Leu Pro Ser Val Asp Tyr Ala Ile Cys145 150 155
160Ser Ser Ser Ser Tyr Trp Gly Ser Thr Val Lys Asn Thr Met Val Cys
165 170 175Ala Gly Gly Asp Gly Val Arg Ser Gly Cys Gln Gly Asp Ser
Gly Gly 180 185 190Pro Leu His Cys Leu Val Asn Gly Lys Tyr Ser Xaa
His Gly Val Thr 195 200 205Ser Phe Val Ser Ser Arg Gly Cys Asn Val
Ser Arg Lys Pro Thr Val 210 215 220Phe Thr Xaa Val Ser Ala Tyr Ile
Ser Trp Ile Asn Asn Val Ile Ala225 230 235 240Ser
Asn95241PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 95Ala Val Val Gly Gly Thr Glu Ala Gly Arg Asn
Ser Trp Pro Ser Gln1 5 10 15Ile Ser Leu Gln Tyr Arg Ser Gly Gly Ser
Xaa Tyr His Thr Cys Gly 20 25 30Gly Thr Leu Ile Arg Gln Asn Trp Val
Xaa Thr Ala Ala His Cys Val 35 40 45Asp Tyr Gln Lys Thr Phe Arg Val
Val Ala Gly Asp His Asn Leu Ser 50 55 60Gln Asn Asp Gly Thr Glu Gln
Tyr Val Ser Val Gln Lys Ile Val Val65 70 75 80His Pro Tyr Trp Asn
Ser Asp Asn Val Ala Ala Gly Tyr Asp Ile Ala 85 90 95Leu Leu Arg Leu
Ala Gln Ser Val Thr Leu Asn Ser Tyr Val Gln Leu 100 105 110Gly Val
Leu Pro Gln Glu Gly Ala Ile Leu Ala Asn Asn Ser Pro Cys 115 120
125Tyr Ile Thr Gly Trp Gly Lys Thr Lys Thr Asn Gly Gln Leu Ala Gln
130 135 140Thr Leu Gln Gln Ala Tyr Leu Pro Ser Val Asp Tyr Ala Ile
Cys Ser145 150 155 160Ser Ser Ser Tyr Trp Gly Ser Thr Val Lys Asn
Thr Met Val Cys Ala 165 170 175Gly Gly Asp Gly Val Arg Ser Gly Cys
Gln Gly Asp Ser Gly Gly Pro 180 185 190Leu His Cys Leu Val Asn Gly
Lys Tyr Ser Xaa His Gly Val Thr Ser 195 200 205Phe Val Ser Ser Arg
Gly Cys Asn Val Ser Arg Lys Pro Thr Val Phe 210 215 220Thr Xaa Val
Ser Ala Tyr Ile Ser Trp Ile Asn Asn Val Ile Ala Ser225 230 235
240Asn9689PRTUnknownDescription of Unknown Yeast alpha-mating
factor signal peptide, propeptide, and spacer sequence 1 96Met Arg
Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala
Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25
30Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu
Leu 50 55 60Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu
Gly Val65 70 75 80Ser Leu Glu Lys Arg Glu Ala Glu Ala
859783PRTUnknownDescription of Unknown Yeast alpha-mating factor
signal peptide and propeptide sequence 2 97Met Arg Phe Pro Ser Ile
Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Ala Pro
Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30Ile Pro Ala Glu
Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35 40 45Asp Val Ala
Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60Phe Ile
Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val65 70 75
80Ser Leu Glu98250PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 98Thr Xaa Asp Leu Pro Glu Thr Asn
Pro Ala Val Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro
Ser Gln Ile Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Xaa Tyr His
Thr Cys Gly Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Xaa Thr Ala
Ala His Cys Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly
Asp His Asn Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val
Ser Val Gln Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn
Val Ala Ala Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105
110Val Thr Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly
115 120 125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp
Gly Lys 130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln
Gln Ala Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser
Ser Ser Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val
Cys Ala Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp
Ser Gly Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser
Xaa His Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn
Val Ser Arg Lys Pro Thr Val Phe Thr Xaa Val Ser Ala Tyr Ile225 230
235 240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
25099250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 99Thr Xaa Asp Leu Pro Glu Thr Asn His Ala Val
Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Xaa Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Xaa Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val Gln
Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala Ala
Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr
Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Xaa Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
250100250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 100Thr Xaa Asp Leu Pro Glu Thr Lys Pro Ala
Val Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln
Ile Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Xaa Tyr His Thr Cys
Gly Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Xaa Thr Ala Ala His
Cys Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His
Asn Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val
Gln Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala
Ala Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val
Thr Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Xaa Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
250101250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 101Thr Xaa Asp Leu Pro Glu Thr His Pro Ala
Val Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln
Ile Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Xaa Tyr His Thr Cys
Gly Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Xaa Thr Ala Ala His
Cys Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His
Asn Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val
Gln Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala
Ala Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val
Thr Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Xaa Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
250102250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 102Thr Xaa Asp Leu Pro Glu His Asn Pro Ala
Val Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln
Ile Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Xaa Tyr His Thr Cys
Gly Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Xaa Thr Ala Ala His
Cys Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His
Asn Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val
Gln Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala
Ala Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val
Thr Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Xaa Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245
250103250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 103Thr Xaa Asp Leu Pro His Thr Asn Pro Ala
Val Val Gly Gly Thr Glu1 5 10 15Ala Gly Arg Asn Ser Trp Pro Ser Gln
Ile Ser Leu Gln Tyr Arg Ser 20 25 30Gly Gly Ser Xaa Tyr His Thr Cys
Gly Gly Thr Leu Ile Arg Gln Asn 35 40 45Trp Val Xaa Thr Ala Ala His
Cys Val Asp Tyr Gln Lys Thr Phe Arg 50 55 60Val Val Ala Gly Asp His
Asn Leu Ser Gln Asn Asp Gly Thr Glu Gln65 70 75 80Tyr Val Ser Val
Gln Lys Ile Val Val His Pro Tyr Trp Asn Ser Asp 85 90 95Asn Val Ala
Ala Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val
Thr Leu Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly 115 120
125Ala Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys
130 135 140Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala
Tyr Leu145 150 155 160Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser
Ser Tyr Trp Gly Ser 165 170 175Thr Val Lys Asn Thr Met Val Cys Ala
Gly Gly Asp Gly Val Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly
Gly Pro Leu His Cys Leu Val Asn Gly 195 200 205Lys Tyr Ser Xaa His
Gly Val Thr Ser Phe Val Ser Ser Arg Gly Cys 210 215 220Asn Val Ser
Arg Lys Pro Thr Val Phe Thr Xaa Val Ser Ala Tyr Ile225 230 235
240Ser Trp Ile Asn Asn Val Ile Ala Ser Asn 245 250104242PRTHomo
sapiensMOD_RES(28)..(28)Trp or Arg 104Ala Arg Val Val Gly Gly Thr
Glu Ala Gly Arg Asn Ser Trp Pro Ser1
5 10 15Gln Ile Ser Leu Gln Tyr Arg Ser Gly Gly Ser Xaa Tyr His Thr
Cys 20 25 30Gly Gly Thr Leu Ile Arg Gln Asn Trp Val Xaa Thr Ala Ala
His Cys 35 40 45Val Asp Tyr Gln Lys Thr Phe Arg Val Val Ala Gly Asp
His Asn Leu 50 55 60Ser Gln Asn Asp Gly Thr Glu Gln Tyr Val Ser Val
Gln Lys Ile Val65 70 75 80Val His Pro Tyr Trp Asn Ser Asp Asn Val
Ala Ala Gly Tyr Asp Ile 85 90 95Ala Leu Leu Arg Leu Ala Gln Ser Val
Thr Leu Asn Ser Tyr Val Gln 100 105 110Leu Gly Val Leu Pro Gln Glu
Gly Ala Ile Leu Ala Asn Asn Ser Pro 115 120 125Cys Tyr Ile Thr Gly
Trp Gly Lys Thr Lys Thr Asn Gly Gln Leu Ala 130 135 140Gln Thr Leu
Gln Gln Ala Tyr Leu Pro Ser Val Asp Tyr Ala Ile Cys145 150 155
160Ser Ser Ser Ser Tyr Trp Gly Ser Thr Val Lys Asn Thr Met Val Cys
165 170 175Ala Gly Gly Asp Gly Val Arg Ser Gly Cys Gln Gly Asp Ser
Gly Gly 180 185 190Pro Leu His Cys Leu Val Asn Gly Lys Tyr Ser Xaa
His Gly Val Thr 195 200 205Ser Phe Val Ser Ser Arg Gly Cys Asn Val
Ser Arg Lys Pro Thr Val 210 215 220Phe Thr Xaa Val Ser Ala Tyr Ile
Ser Trp Ile Asn Asn Val Ile Ala225 230 235 240Ser Asn105241PRTHomo
sapiensMOD_RES(27)..(27)Trp or Arg 105Arg Val Val Gly Gly Thr Glu
Ala Gly Arg Asn Ser Trp Pro Ser Gln1 5 10 15Ile Ser Leu Gln Tyr Arg
Ser Gly Gly Ser Xaa Tyr His Thr Cys Gly 20 25 30Gly Thr Leu Ile Arg
Gln Asn Trp Val Xaa Thr Ala Ala His Cys Val 35 40 45Asp Tyr Gln Lys
Thr Phe Arg Val Val Ala Gly Asp His Asn Leu Ser 50 55 60Gln Asn Asp
Gly Thr Glu Gln Tyr Val Ser Val Gln Lys Ile Val Val65 70 75 80His
Pro Tyr Trp Asn Ser Asp Asn Val Ala Ala Gly Tyr Asp Ile Ala 85 90
95Leu Leu Arg Leu Ala Gln Ser Val Thr Leu Asn Ser Tyr Val Gln Leu
100 105 110Gly Val Leu Pro Gln Glu Gly Ala Ile Leu Ala Asn Asn Ser
Pro Cys 115 120 125Tyr Ile Thr Gly Trp Gly Lys Thr Lys Thr Asn Gly
Gln Leu Ala Gln 130 135 140Thr Leu Gln Gln Ala Tyr Leu Pro Ser Val
Asp Tyr Ala Ile Cys Ser145 150 155 160Ser Ser Ser Tyr Trp Gly Ser
Thr Val Lys Asn Thr Met Val Cys Ala 165 170 175Gly Gly Asp Gly Val
Arg Ser Gly Cys Gln Gly Asp Ser Gly Gly Pro 180 185 190Leu His Cys
Leu Val Asn Gly Lys Tyr Ser Xaa His Gly Val Thr Ser 195 200 205Phe
Val Ser Ser Arg Gly Cys Asn Val Ser Arg Lys Pro Thr Val Phe 210 215
220Thr Xaa Val Ser Ala Tyr Ile Ser Trp Ile Asn Asn Val Ile Ala
Ser225 230 235 240Asn106236PRTHomo sapiensMOD_RES(22)..(22)Trp or
Arg 106Thr Glu Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile Ser Leu Gln
Tyr1 5 10 15Arg Ser Gly Gly Ser Xaa Tyr His Thr Cys Gly Gly Thr Leu
Ile Arg 20 25 30Gln Asn Trp Val Xaa Thr Ala Ala His Cys Val Asp Tyr
Gln Lys Thr 35 40 45Phe Arg Val Val Ala Gly Asp His Asn Leu Ser Gln
Asn Asp Gly Thr 50 55 60Glu Gln Tyr Val Ser Val Gln Lys Ile Val Val
His Pro Tyr Trp Asn65 70 75 80Ser Asp Asn Val Ala Ala Gly Tyr Asp
Ile Ala Leu Leu Arg Leu Ala 85 90 95Gln Ser Val Thr Leu Asn Ser Tyr
Val Gln Leu Gly Val Leu Pro Gln 100 105 110Glu Gly Ala Ile Leu Ala
Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp 115 120 125Gly Lys Thr Lys
Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala 130 135 140Tyr Leu
Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser Ser Tyr Trp145 150 155
160Gly Ser Thr Val Lys Asn Thr Met Val Cys Ala Gly Gly Asp Gly Val
165 170 175Arg Ser Gly Cys Gln Gly Asp Ser Gly Gly Pro Leu His Cys
Leu Val 180 185 190Asn Gly Lys Tyr Ser Xaa His Gly Val Thr Ser Phe
Val Ser Ser Arg 195 200 205Gly Cys Asn Val Ser Arg Lys Pro Thr Val
Phe Thr Xaa Val Ser Ala 210 215 220Tyr Ile Ser Trp Ile Asn Asn Val
Ile Ala Ser Asn225 230 235107234PRTHomo sapiensMOD_RES(20)..(20)Any
amino acid 107Ala Gly Arg Asn Ser Trp Pro Ser Gln Ile Ser Leu Gln
Tyr Arg Ser1 5 10 15Gly Gly Ser Xaa Tyr His Thr Cys Gly Gly Thr Leu
Ile Arg Gln Asn 20 25 30Trp Val Xaa Thr Ala Ala His Cys Val Asp Tyr
Gln Lys Thr Phe Arg 35 40 45Val Val Ala Gly Asp His Asn Leu Ser Gln
Asn Asp Gly Thr Glu Gln 50 55 60Tyr Val Ser Val Gln Lys Ile Val Val
His Pro Tyr Trp Asn Ser Asp65 70 75 80Asn Val Ala Ala Gly Tyr Asp
Ile Ala Leu Leu Arg Leu Ala Gln Ser 85 90 95Val Thr Leu Asn Ser Tyr
Val Gln Leu Gly Val Leu Pro Gln Glu Gly 100 105 110Ala Ile Leu Ala
Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys 115 120 125Thr Lys
Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala Tyr Leu 130 135
140Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser Ser Tyr Trp Gly
Ser145 150 155 160Thr Val Lys Asn Thr Met Val Cys Ala Gly Gly Asp
Gly Val Arg Ser 165 170 175Gly Cys Gln Gly Asp Ser Gly Gly Pro Leu
His Cys Leu Val Asn Gly 180 185 190Lys Tyr Ser Xaa His Gly Val Thr
Ser Phe Val Ser Ser Arg Gly Cys 195 200 205Asn Val Ser Arg Lys Pro
Thr Val Phe Thr Xaa Val Ser Ala Tyr Ile 210 215 220Ser Trp Ile Asn
Asn Val Ile Ala Ser Asn225 230108231PRTHomo
sapiensMOD_RES(17)..(17)Trp or Arg 108Asn Ser Trp Pro Ser Gln Ile
Ser Leu Gln Tyr Arg Ser Gly Gly Ser1 5 10 15Xaa Tyr His Thr Cys Gly
Gly Thr Leu Ile Arg Gln Asn Trp Val Xaa 20 25 30Thr Ala Ala His Cys
Val Asp Tyr Gln Lys Thr Phe Arg Val Val Ala 35 40 45Gly Asp His Asn
Leu Ser Gln Asn Asp Gly Thr Glu Gln Tyr Val Ser 50 55 60Val Gln Lys
Ile Val Val His Pro Tyr Trp Asn Ser Asp Asn Val Ala65 70 75 80Ala
Gly Tyr Asp Ile Ala Leu Leu Arg Leu Ala Gln Ser Val Thr Leu 85 90
95Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln Glu Gly Ala Ile Leu
100 105 110Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Lys Thr
Lys Thr 115 120 125Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala Tyr
Leu Pro Ser Val 130 135 140Asp Tyr Ala Ile Cys Ser Ser Ser Ser Tyr
Trp Gly Ser Thr Val Lys145 150 155 160Asn Thr Met Val Cys Ala Gly
Gly Asp Gly Val Arg Ser Gly Cys Gln 165 170 175Gly Asp Ser Gly Gly
Pro Leu His Cys Leu Val Asn Gly Lys Tyr Ser 180 185 190Xaa His Gly
Val Thr Ser Phe Val Ser Ser Arg Gly Cys Asn Val Ser 195 200 205Arg
Lys Pro Thr Val Phe Thr Xaa Val Ser Ala Tyr Ile Ser Trp Ile 210 215
220Asn Asn Val Ile Ala Ser Asn225 230109781DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
109gaattcagta ctcaggacct tccggaaacc aatgcccggg tagtcggagg
gactgaggcc 60gggaggaact cctggccctc tcagatttcc ctccagtacc ggtctggagg
ttcctggtat 120cacacctgtg gagggaccct tatcagacag aactgggtga
tgacagctgc acactgcgtg 180gattaccaga agactttccg cgtggtggct
ggagaccata acctgagcca gaatgatggc 240actgagcagt acgtgagtgt
gcagaagatc gtggtgcatc catactggaa cagcgataac 300gtggctgcag
gctatgacat cgccctgctg cgcctggccc agagcgttac cctcaatagc
360tatgtccagc tgggtgttct gccccaggag ggagccatcc tggctaacaa
cagtccctgc 420tacatcacag gctggggcaa gaccaagacc aatgggcagc
tggcccagac cttgcagcag 480gcttacctgc cctctgtgga ctatgccatc
tgctccagct cctcctactg gggctccact 540gtgaagaaca ctatggtgtg
tgctggtgga gatggagttc gctctggatg tcagggtgac 600tctgggggcc
ccctccattg cttggtgaat ggcaagtatt ctcttcatgg agtgaccagc
660tttgtgtcca gccggggctg taatgtctct agaaagccta cagtcttcac
acgggtctct 720gcttacatct cctggataaa taatgtcatc gcctccaact
gataagcttg gatccgtcga 780c 781110213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
110Met Lys Arg Ile Leu Ala Ile His Gln Ala Met Glu Gly Ala Pro Arg1
5 10 15Val Thr Leu Thr Ser Arg Ala Asn Ser Ile Ser Thr Ser Thr His
His 20 25 30Ser Val Leu His Ser Gly Ala Pro Val Gly Gly Ala Gly Ala
Asp Gly 35 40 45Ile Val His Arg Gly Gln Val Ser Leu Leu Gln Gly Leu
Gly Gln Leu 50 55 60Pro Ile Gly Leu Gly Leu Ala Pro Ala Cys Asp Val
Ala Gly Thr Val65 70 75 80Val Ser Gln Asp Gly Ser Leu Leu Gly Gln
Asn Thr Gln Leu Asp Ile 85 90 95Ala Ile Glu Gly Asn Ala Leu Gly Gln
Ala Gln Gln Gly Asp Val Ile 100 105 110Ala Cys Ser His Val Ile Ala
Val Pro Val Trp Met His His Asp Leu 115 120 125Leu His Thr His Val
Leu Leu Ser Ala Ile Ile Leu Ala Gln Val Met 130 135 140Val Ser Ser
His His Ala Glu Ser Leu Leu Val Ile His Ala Val Cys145 150 155
160Ser Cys His His Pro Val Leu Ser Asp Lys Gly Pro Ser Thr Gly Val
165 170 175Ile Pro Gly Thr Ser Arg Pro Val Leu Glu Gly Asn Leu Arg
Gly Pro 180 185 190Gly Val Pro Pro Gly Leu Ser Pro Ser Asp Tyr Pro
Gly Ile Gly Phe 195 200 205Arg Lys Val Leu Ser
210111105DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 111act att gcc agc att gct gct aaa gaa gaa
ggg gta tct ctc gag aaa 48Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu
Gly Val Ser Leu Glu Lys1 5 10 15aga gag gct gaa gct act cag gac ctt
ccg gaa acc aat gcc cgg gta 96Arg Glu Ala Glu Ala Thr Gln Asp Leu
Pro Glu Thr Asn Ala Arg Val 20 25 30gtc ggg ggg 105Val Gly Gly
3511235PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 112Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu
Gly Val Ser Leu Glu Lys1 5 10 15Arg Glu Ala Glu Ala Thr Gln Asp Leu
Pro Glu Thr Asn Ala Arg Val 20 25 30Val Gly Gly
35113768DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 113ccgcggaccc aggactttcc agaaaccaac
gcccgggtag ttggagggac cgaggctcag 60aggaattctt ggccatctca gatttccctc
cagtaccggt ctggaagttc gtgggctcac 120acctgtggag ggaccctcat
caggcagaac tgggtgatga cagccgctca ctgcgtggac 180agagagttga
ccttccgtgt ggtggttgga gagcacaacc tgaaccagaa cgatggcacc
240gagcagtacg tgggggtgca gaagatcgtg gtgcatccct actggaacac
cgacgacgtg 300gctgcaggct atgacatcgc cctgctgcgc ctggcccaga
gtgtaaccct caacagctac 360gtccagctgg gtgttctgcc aagggctggg
accatcctgg ctaacaacag tccctgctac 420atcacagggt ggggcctgac
caggaccaat gggcagctgg cccagaccct gcagcaggct 480tacctgccca
ccgtggacta cgccatctgc tccagctcct cgtactgggg ctccaccgtg
540aagaacagca tggtgtgcgc cggaggggac ggagttcgct ctggatgtca
gggtgattct 600gggggccccc ttcattgctt ggtgaatggt cagtatgctg
tccacggtgt aaccagcttc 660gtgtcccgcc tgggctgtaa tgtcaccagg
aagcccacag tcttcaccag ggtctctgct 720tacatctctt ggataaataa
cgtcattgcc agcaactgat aatctaga 768114250PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
114Thr Gln Asp Phe Pro Glu Thr Asn Ala Arg Val Val Gly Gly Thr Glu1
5 10 15Ala Gln Arg Asn Ser Trp Pro Ser Gln Ile Ser Leu Gln Tyr Arg
Ser 20 25 30Gly Ser Ser Trp Ala His Thr Cys Gly Gly Thr Leu Ile Arg
Gln Asn 35 40 45Trp Val Met Thr Ala Ala His Cys Val Asp Arg Glu Leu
Thr Phe Arg 50 55 60 Val Val Val Gly Glu His Asn Leu Asn Gln Asn
Asp Gly Thr Glu Gln65 70 75 80Tyr Val Gly Val Gln Lys Ile Val Val
His Pro Tyr Trp Asn Thr Asp 85 90 95Asp Val Ala Ala Gly Tyr Asp Ile
Ala Leu Leu Arg Leu Ala Gln Ser 100 105 110Val Thr Leu Asn Ser Tyr
Val Gln Leu Gly Val Leu Pro Arg Ala Gly 115 120 125Thr Ile Leu Ala
Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp Gly Leu 130 135 140Thr Arg
Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala Tyr Leu145 150 155
160Pro Thr Val Asp Tyr Ala Ile Cys Ser Ser Ser Ser Tyr Trp Gly Ser
165 170 175Thr Val Lys Asn Ser Met Val Cys Ala Gly Gly Asp Gly Val
Arg Ser 180 185 190Gly Cys Gln Gly Asp Ser Gly Gly Pro Leu His Cys
Leu Val Asn Gly 195 200 205Gln Tyr Ala Val His Gly Val Thr Ser Phe
Val Ser Arg Leu Gly Cys 210 215 220Asn Val Thr Arg Lys Pro Thr Val
Phe Thr Arg Val Ser Ala Tyr Ile225 230 235 240Ser Trp Ile Asn Asn
Val Ile Ala Ser Asn 245 25011510PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 115Phe Pro Glu Thr Asn Ala
Arg Val Val Gly1 5 1011610PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 116Phe Pro Glu Thr Asn Ala
Ala Val Val Gly1 5 1011710PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 117Leu Pro His Thr Asn Pro
Ala Val Val Gly1 5 1011810PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 118Phe Pro Glu Thr Asn His
Ala Val Val Gly1 5 101193PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 119Xaa Xaa
Xaa11205PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 120Xaa Xaa Xaa Xaa Xaa1 512110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 121Thr
Xaa Asp Leu Pro Xaa Xaa Xaa Xaa Xaa1 5 1012213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 122Thr
Xaa Asp Leu Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
101238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 123Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
51243PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 124Xaa Xaa Xaa11258PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 125Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 51265PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 126Xaa Xaa Xaa Xaa Xaa1
51274PRTHomo sapiens 127Val Val Gly Gly11286PRTHomo sapiens 128Val
Val Gly Gly Thr Glu1 51299PRTHomo sapiens 129Val Val Gly Gly Thr
Glu Ala Gly Arg1 5130781DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 130gaattcagt act cag
gac ctt ccg gaa acc aat gcc cgg gta gtc gga ggg 51 Thr Gln Asp Leu
Pro Glu Thr Asn Ala Arg Val Val Gly Gly 1 5 10act gag gcc ggg agg
aac tcc tgg ccc tct cag att tcc ctc cag tac 99Thr Glu Ala Gly Arg
Asn Ser Trp Pro Ser Gln Ile Ser Leu Gln Tyr15 20 25 30cgg tct gga
ggt tcc tgg tat cac acc tgt gga ggg acc ctt atc aga 147Arg Ser Gly
Gly Ser Trp Tyr His Thr Cys Gly Gly Thr Leu Ile Arg 35 40 45cag aac
tgg gtg atg aca gct gca cac tgc gtg gat tac cag aag act 195Gln Asn
Trp Val Met Thr Ala Ala His Cys Val
Asp Tyr Gln Lys Thr 50 55 60ttc cgc gtg gtg gct gga gac cat aac ctg
agc cag aat gat ggc act 243Phe Arg Val Val Ala Gly Asp His Asn Leu
Ser Gln Asn Asp Gly Thr 65 70 75gag cag tac gtg agt gtg cag aag atc
gtg gtg cat cca tac tgg aac 291Glu Gln Tyr Val Ser Val Gln Lys Ile
Val Val His Pro Tyr Trp Asn 80 85 90agc gat aac gtg gct gca ggc tat
gac atc gcc ctg ctg cgc ctg gcc 339Ser Asp Asn Val Ala Ala Gly Tyr
Asp Ile Ala Leu Leu Arg Leu Ala95 100 105 110cag agc gtt acc ctc
aat agc tat gtc cag ctg ggt gtt ctg ccc cag 387Gln Ser Val Thr Leu
Asn Ser Tyr Val Gln Leu Gly Val Leu Pro Gln 115 120 125gag gga gcc
atc ctg gct aac aac agt ccc tgc tac atc aca ggc tgg 435Glu Gly Ala
Ile Leu Ala Asn Asn Ser Pro Cys Tyr Ile Thr Gly Trp 130 135 140ggc
aag acc aag acc aat ggg cag ctg gcc cag acc ttg cag cag gct 483Gly
Lys Thr Lys Thr Asn Gly Gln Leu Ala Gln Thr Leu Gln Gln Ala 145 150
155tac ctg ccc tct gtg gac tat gcc atc tgc tcc agc tcc tcc tac tgg
531Tyr Leu Pro Ser Val Asp Tyr Ala Ile Cys Ser Ser Ser Ser Tyr Trp
160 165 170ggc tcc act gtg aag aac act atg gtg tgt gct ggt gga gat
gga gtt 579Gly Ser Thr Val Lys Asn Thr Met Val Cys Ala Gly Gly Asp
Gly Val175 180 185 190cgc tct gga tgt cag ggt gac tct ggg ggc ccc
ctc cat tgc ttg gtg 627Arg Ser Gly Cys Gln Gly Asp Ser Gly Gly Pro
Leu His Cys Leu Val 195 200 205aat ggc aag tat tct ctt cat gga gtg
acc agc ttt gtg tcc agc cgg 675Asn Gly Lys Tyr Ser Leu His Gly Val
Thr Ser Phe Val Ser Ser Arg 210 215 220ggc tgt aat gtc tct aga aag
cct aca gtc ttc aca cgg gtc tct gct 723Gly Cys Asn Val Ser Arg Lys
Pro Thr Val Phe Thr Arg Val Ser Ala 225 230 235tac atc tcc tgg ata
aat aat gtc atc gcc tcc aac tgataagctt 769Tyr Ile Ser Trp Ile Asn
Asn Val Ile Ala Ser Asn 240 245 250ggatccgtcg ac
7811318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 131Thr Asn Ala Arg Val Val Gly Gly1 5
* * * * *
References