U.S. patent application number 10/205270 was filed with the patent office on 2003-05-01 for method for making acylated polypeptides.
Invention is credited to Balschmidt, Per, Diers, Ivan, Egel-Mitani, Michi, Hoeg-Jensen, Thomas, Markussen, Jan.
Application Number | 20030082671 10/205270 |
Document ID | / |
Family ID | 27222523 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030082671 |
Kind Code |
A1 |
Hoeg-Jensen, Thomas ; et
al. |
May 1, 2003 |
Method for making acylated polypeptides
Abstract
The present invention related to a method of producing
polypeptides in transformed host cells by expressing a precursor
molecule of the desired polypeptide which are to be acylated in a
subsequent in vitro step. The invention is also related to
DNA-sequences, vectors and transformed host cells for use in the
claimed method. Further, the present invention is related to
certain precursors of the desired polypeptides and certain
acylation methods. The invention provides a method for making
polypeptides being preferentially acylated in certain lysine
.epsilon.-amino groups.
Inventors: |
Hoeg-Jensen, Thomas;
(Klampenborg, DK) ; Egel-Mitani, Michi; (Vedbaek,
DK) ; Balschmidt, Per; (Espergaerde, DK) ;
Markussen, Jan; (Herlev, DK) ; Diers, Ivan;
(Vaerlose, DK) |
Correspondence
Address: |
Reza Green, Esq.
Novo Nordisk of North America, Inc.
Suite 6400
405 Lexington Avenue
New York
NY
10174-6400
US
|
Family ID: |
27222523 |
Appl. No.: |
10/205270 |
Filed: |
July 24, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60310952 |
Aug 8, 2001 |
|
|
|
Current U.S.
Class: |
435/68.1 ;
435/69.1 |
Current CPC
Class: |
C07K 1/006 20130101;
C07K 1/1077 20130101; C07K 14/605 20130101 |
Class at
Publication: |
435/68.1 ;
435/69.1 |
International
Class: |
C12P 021/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 24, 2001 |
DK |
PA 2001 01140 |
Claims
What is claimed is:
1. A method for making an acylated polypeptide, wherein said
polypeptide comprises at least one lysine residue that is acylated
on its .epsilon.-amino group, said method comprising: (i)
expressing in a suitable host cell a precursor molecule of the
polypeptide, wherein said precursor comprises said polypeptide and
an N-terminal extension, wherein said N-terminal extension is
capable of protecting the desired polypeptide against proteolytic
degradation and has a cleavage site different from Lys positioned
at its C-terminal; (ii) acylating the .epsilon.-amino group of at
least one lysine residue in the desired polypeptide; and (iii)
removing the N-terminal extension by chemical and/or enzymatic
cleavage.
2. A method according to claim 1, wherein the acylating step is
conducted after the removing step.
3. A method according to claim 2, wherein the polypeptide is
monoacylated.
4. A method according to claim 1, wherein the N-terminal extension
is up to 15 amino acid residues in length.
5. A method according to claim 4, wherein the N-terminal extension
is 3-12 amino acid residues in length.
6. A method according to claim 1, wherein the polypeptide belongs
to the GRF (growth hormone releasing factor) family of peptides
having a His or Tyr in the N-terminal position and Ser, Ala or Gly
in the next position.
7. A method according to claim 6, wherein the polypeptide has a
His-Ala, His-Gly, His-Ser or Tyr-Ala as the N-terminal
sequence.
8. A method according to claim 1, wherein the polypeptide is GLP-1
or GLP-2 or a GLP-1 or GLP-2 analogue.
9. A method according to claim 8, wherein the polypeptide is
Arg.sup.34GLP1.sub.(7-37) acylated in position Lys.sup.26.
10. A method according to claim 1, wherein the cleavage site in the
N-terminal extension is selected from the group consisting of Met,
Asn, Pro, Gln, Cys and Arg-Arg.
11. A method according to claim 1, wherein the N-terminal extension
comprises a Glu-Glu sequence at the N-terminal end.
12. A method according to claim 1, wherein the N-terminal extension
has the sequenceX.sub.n - - - X.sub.1--Ywherein X.sub.n - - -
X.sub.1 is a peptide sequence of from 1-14 amino acid residues in
length, the sequence X.sub.n - - - X.sub.1--Y having the function
of a) protecting the expressed polypeptide from endoproteolytic
cleavage, b) preventing acylation of the N-terminal end of the
polypeptide and c) preventing precipitation caused by fibrillation
during fermentation and downstream separation and purification
steps; and Y is Met; Asn, Pro, Gln, Cys or Arg-Arg.
13. A method according to claim 12, wherein X.sub.n - - - X.sub.1
is a peptide sequence of from 2-14 amino acid residues in
length.
14. A method according to claim 12, wherein X.sub.n - - - X.sub.1
is a peptide sequence of from 3-12 amino acid residues in
length.
15. A method according to claim 1, wherein the N-terminal extension
is selected from the group consisting of Glu-Glu-Met;
Glu-Glu-Ala-Glu-Met(SEQ ID NO:1); Glu-Glu-Ala-Glu-Asn(SEQ ID NO:2);
Glu-Glu-Ala-Glu-Arg-Arg(SEQ ID NO:3); Gln; Glu-Pro-Gln(SEQ ID
NO:4); Glu-Ala-Gln; Glu-Ala-Glu-Ala-Gln(SEQ ID NO:5);
Glu-Ala-Glu-Ala-Glu-Ala-Gl- n(SEQ ID NO:6);
Glu-Glu-Gly-Cys-Thr-Ser-Ile-Cys(SEQ ID NO:7);
Glu-His-Gly-Cys-Thr-Ser-Ile-Cys(SEQ ID NO:8);
Glu-Glu-Ala-Arg-Met(SEQ ID NO:9); Glu-Glu-Arg-Asn(SEQ ID NO:10);
Glu-Glu-Ala-Glu-Asn(SEQ ID NO:11); Glu-Glu-Arg-Ala-Arg-Arg(SEQ ID
NO:12); Glu-Glu-Ala-Glu-Pro(SEQ ID NO:13); Glu-Glu-Gly-Glu-Pro(SEQ
ID NO:14); Glu-Glu-Ala-Glu-Cys(SEQ ID NO:15); and
Glu-Glu-Ile-Glu-Gly-Arg(SEQ ID NO:16).
16. A method according to claim 1, wherein the host cell is a yeast
cell.
17. A method according to claim 16, wherein the yeast cell is a
Saccharomyces cerevisiae cell.
18. A precursor of a polypeptide, said precursor having the
formulaN-terminal extension-Y-*polypeptide*wherein Y is Met, Asn,
Pro, Gin, Cys or Arg-Arg, the N-terminal extension has 1-14 amino
acid residues and *polypeptide* is the remaining sequence of the
polypeptide.
19. A precursor according to claim 18 having the formulaN-terminal
extension-Y.sub.1--Y.sub.2- Y.sub.3- *polypeptide*wherein Y.sub.1
is Met, Asn, Pro, Gln, Cys or Arg-Arg; Y.sub.2 is His or Tyr,
Y.sub.3 is Ala, Ser or Gly, the N-terminal extension has 1-14 amino
acid residues and *polypeptide* is the remaining part of desired
polypeptide.
20. A precursor according to claim 19, wherein the polypeptide is
GLP-1 or GLP-2 or a GLP-1 or GLP-2 analogue.
21. A precursor according to claim 20, wherein the polypeptide is
Arg.sup.34GLP1.sub.(7-37) acylated in position Lys.sup.26.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. 119 of
Danish application no. PA 2001 01140 filed on Jul. 24, 2001, and
U.S. application No. 60/310,952 filed on Aug. 8, 2001, the contents
of which are fully incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention is related to a method of producing
acylated proteins or polypeptides by expressing certain precursors
of the desired polypeptide that protects the expressed polypeptide
against proteolytic degradation within the host cell. The invention
is also related to DNA-sequences, vectors and transformed host
cells for use in the claimed method. Furthermore, the present
invention is related to certain precursors of the desired
polypeptides and an acylation method for acylation method for
acylation of one or more lysine residues in the desired
polypeptide.
BACKGROUND OF THE INVENTION
[0003] Recombinant DNA technology has enabled expression of foreign
(heterologous) polypeptides in microbial and other host cells. In
yeast expression of heterologous polypeptides after transformation
of yeast cells with suitable expression vectors comprising DNA
sequences coding for said polypeptides has been successful for many
species of polypeptides, such as insulin and insulin precursors,
glucagon, glucagon like peptides and analogues thereof.
[0004] A common problem with expression of proteins or polypeptides
of a limited size in a recombinant host is, however, proteolytic
degradation of the expressed product by proteolytic enzymes
produced by the host organism.
[0005] Thus, the isolated product may be a heterogeneous mixture of
species of the desired polypeptide having different amino acid
chain lengths. Another problem encountered in production of
heterologous polypeptides in yeast may be low yield, presumably due
to proteolytic processing both in intracellular compartments and at
the plasma membrane caused by aberrant processing at internal sites
in the polypeptide. Yeast contains a number of proteases used for
processing yeast proteins e.g. Kex2p and Yps1p which cleave at the
C-terminal side of a dibasic amino acid sequence, and the
carboxypeptidase Kex1p which digests remaining basic amino acids
after the endoproteolytic digestion by Kex2p, and Ste13p or Dap2p
which cleave at X-Ala or X-Pro.
[0006] Some polypeptides, e.g. polypeptides having from about 10 to
about 100 amino acids chains and none or a few disulphide bonds
and/or are rich in basic amino acids, such as .beta.-endorphine,
glucagon and glucagon like peptides may be especially susceptible
to intracellular and extracellular proteolytic degradation when
expressed in a transformed host cell due to their short-chain open
and non-disulfide stabilized structure resulting in an
inhomogeneous product which may be proteolytically degraded in the
N- and C-terminal ends as well as endoproteolytically degraded.
[0007] Furthermore, N-terminal cleavage of expressed polypeptides
by host cell produced enzymes may cause decreased yield of a
desired product with correct N-terminal if the N-terminal of the
expressed product constitutes a cleavage site for endogenous
enzymes. In yeast for example the enzyme Ste13p cleaves at X-Ala or
X-Pro, where X can be any amino acid residue. Thus, polypeptides
with an Ala or Pro residue as the second residue from the
N-terminal end may be cleaved at the N-terminal end and the
recovered polypeptide may be a mixture of different degradation
products complicating the recovery process and reducing the overall
yield.
[0008] Furthermore, small polypeptides with little tertiary
structure and low content of .alpha.-helixes may have a higher
tendency to form .beta.-sheets that stack on each other and form
fibrils during fermentation and down stream separation and
purification steps in large scale production. Formation of fibrils
may cause unwanted precipitation with loss of the desired product.
Fibrillation may be prevented by treatment at high pH. However,
such alkaline treatment is pretty harsh to the product and may
cause unwanted formation of D-amino acids residues.
[0009] Human GLP-1 is a 37 amino acid residue peptide originating
from preproglucagon which is synthesised in the L-cells in the
distal ileum, in the pancreas and in the brain. Processing of
preproglucagon to give GLP-1.sub.(7-36)amide, GLP-1.sub.(7-37) and
GLP-2 occurs mainly in the L-cells. Both GLP-1 and GLP-2 have an
Ala as the second amino acid residue from the N-terminal end and
are thus prone for N-terminal cleavage when expressed in a host
organism such as yeast.
[0010] Introduction of lipophilic acyl groups in naturally
occurring peptides or analogues thereof has shown to lead to
acylated peptides which have a protracted profile relative to the
native peptide or unmodified analogues. The present invention
provides a method for ensuring preferential acylation at the
desired position in the polypeptide in question as it will appear
from the following.
SUMMARY OF THE INVENTION
[0011] In one aspect the present invention is related to a method
for making a polypeptide comprising at least one lysine residue
being acylated in its .epsilon.-amino group, said method comprising
the following steps:
[0012] (i) culturing a host cell comprising a polynucleotide
sequence encoding a precursor molecule of the desired polypeptide,
said precursor molecule comprising an N-terminal extension capable
of protecting the desired polypeptide against proteolytic
degradation, said N-terminal extension comprising a cleavage site
positioned at its C-terminal end for cleavage from the desired
polypeptide, under suitable conditions for expression of said
precursor molecule, said cleavage site being not a Lys residue;
[0013] (ii) separating the expressed precursor from the culture
broth;
[0014] (iii) acylating the .epsilon.-amino group of at least one
lysine residue in the desired polypeptide;
[0015] (iv) removing the N-terminal extension by chemical or
enzymatic cleavage and
[0016] (v) isolating the acylated polypeptide by suitable
means.
DETAILED DESCRIPTION OF THE INVENTION
[0017] In one embodiment of the present invention the order of
steps (ii) to (iv) may be changed. Thus, in one embodiment of the
present invention the acylating step (iii) is conducted after the
removal step (iv) so that the N-terminal extension is removed
before the polypeptide is acylated in the desired position.
[0018] The desired polypeptide may contain more than one lysine
residue as a potential target for acylation but will typically only
contain one lysine residue. Thus, in one embodiment, the desired
polypeptide is monoacylated.
[0019] The N-terminal extension will typically be of up to 15 amino
acids in length and may be from 1-15; 2-15; 3-15; 3-12; 3-10; 3-9;
3-8; 3-7; 3-6; or 3-5 amino acids in length. The amino acids in the
N-terminal extension are selected with a multiple purpose: 1) to
protect the expressed precursor molecule against endoproteolytic
degradation; 2) to avoid acylation at the N-terminal amino acid
residue of the desired polypeptide, i.e. to ensure that acylation
takes place preferentially or only at the wanted position in the
desired polypeptide; and 3) to prevent precipitation caused by
fibrillation during fermentation and down stream processing steps
such as separation and purification in large scale production.
Furthermore, the amino acid residues at both ends of the N-terminal
extension should be selected so as to ensure efficient cleavage of
the N-terminal extension from the desired polypeptide at the
C-terminal end and at the N-terminal end from possible upstream
sequences such as pre- or pre-pro peptides which have the purpose
of ensuring transport of the expressed precursor molecule out of
the host cell and into the culture medium. Finally, the N-terminal
extension may serve as a tag for purification purposes.
[0020] The N-terminal extension is found to be stably attached to
the precursor molecule of the invention during fermentation,
protecting the N-terminal end of the precursor molecule against the
proteolytic enzymes such as Ste13p and/or Dap2p.
[0021] The N-terminal extension is removably attached to the
N-terminal end of the desired polypeptide. Thus, the C-terminal end
of the N-terminal extension will constitute a cleavage site or will
together with amino acid residues at the N-terminal end of the
desired polypeptide constitute a suitable cleavage site. This
cleavage site is different from lysine to avoid acylation of the
precursor molecule at this position.
[0022] Cleavage may be conducted by means of chemicals like
cyanogen bromide (E. Gross: Methods in Enzymlogy XI, 1967, 238-255,
Editor: CHW Hirs, and J P Whitelegge et al, Protein Science 2000,
9, 1618-1630) or hydroxylamine cleaving at the C-terminal side of
Met or Asn. In the case of Asn cleavage is enhanced by the presence
of a Gly N-terminal to the cleavage site (Asn .dwnarw.Gly). The
cleavage can also be effected by specific exoproteases such as a
suitable proteolytic enzyme which is specific for the chosen
cleavage site, cleaving at an N-terminal pyroglutamic acid or
endoproteases like proline endopeptidases (EC 3.4.22.26) cleaving
at the C-terminal side of Pro in polypeptides. Other endoproteases
as trypsins cleave at the C-terminal side of many single
Arg-residues, while other like Factor Xa is more specific and often
cleaves after the sequence Ile-Glu-Gly-Arg. Kex2p or PC1 and
similar enzymes cleave at a dibasic cleavage site such as Arg-Arg.
A mixture of chemical and enzymatic methods can be used if a Cys
residue is placed N-terminally to the polypeptide. The SH-group can
easily be acylated with an amine that is generating a pseudo-lysine
amino acid which can then be cleaved by Achromobacter lyticus
protease I after acylation of the lysine residue in the desired
polypeptide, thereby protecting this site from cleavage.
[0023] In one embodiment, the cleavage site is chosen from the
group consisting of Met, Asn, Pro, Gin, Cys and Arg-Arg.
[0024] According to one embodiment of the present invention, the
desired polypeptide will comprise a Pro, Ala or Ser residue as the
second amino acid residue from the N-terminal end. Such
polypeptides will be especially vulnerable to degradation by
proteolytic enzymes such as Ste13p.
[0025] According to a further embodiment, the desired polypeptide
will have a His-Ala or a His-Pro, His-Ser or a Tyr as the
N-terminal sequence.
[0026] In a more specific embodiment, the N-terminal extension has
the formula
X.sub.n - - - X.sub.1--Y
[0027] wherein X.sub.n - - - X.sub.1 is a peptide sequence of from
1 - 14 amino acid residues in length and Y is Met, Asn, Pro, Gln,
Cys or Arg-Arg, the function of X.sub.n - - - X.sub.1--Y being a)
to protect the expressed polypeptide from endoproteolytic cleavage,
b) to prevent acylation at the N-terminal end of the desired
polypeptide and c) to prevent precipitation caused by fibrillation
during fermentation and down stream separation and purification
steps. The amino acid residues in X.sub.n - - - X.sub.1--Y are
furthermore selected to obtain optimal in vitro cleavage of the
N-terminal extension at its C-terminal end (at Y) and optimal in
vivo cleavage at its N-terminal end (at X.sub.n) at a KEX site from
upstream signal-leader sequences. The amino acid residues in
X.sub.n - - - X.sub.1 may in principle be any amino acid residue
except Lys as long as the peptide sequence fulfils at least one of
the required purposes. However, number two amino acid residue from
the N-terminal end of the extension is preferably not Ala or
Pro.
[0028] In one embodiment X.sub.n - - - X.sub.1 is a peptide
sequence of from 1-15; 2-15; 3-15; 3-12; 3-10; 3-9; 3-8; 3-7; 3-6;
or 3-5 amino acid residues in length. To ensure efficient cleavage
from a pre-pro-sequence at a Kex2 site in a host cell as yeast one
or two of the N-terminal amino acid residues in X.sub.n - - -
X.sub.1 are preferably chosen from Glu and Asp. Glu and/or Asp
positioned at the N-terminal end of the N-terminal extension will
also protect the expressed molecule against proteolytic degradation
in the yeast cell.
[0029] Examples of N-terminal extensions are Glu-Glu-Met;
Glu-Glu-Ala-Glu-Met(SEQ ID NO:1); Glu-Glu-Ala-Glu-Asn(SEQ ID NO:2);
Glu-Glu-Ala-Glu-Arg-Arg(SEQ ID NO:3); Gln; Glu-Pro-Gln(SEQ ID
NO:4); Glu-Ala-Gln; Glu-Ala-Glu-Ala-Gln(SEQ ID NO:5);
Glu-Ala-Glu-Ala-Glu-Ala-Gl- n(SEQ ID NO:6);
Glu-Glu-Gly-Cys-Thr-Ser-Ile-Cys(SEQ ID NO:7);
Glu-His-Gly-Cys-Thr-Ser-Ile-Cys(SEQ ID NO:8);
Glu-Glu-Ala-Arg-Met(SEQ ID NO:9); Glu-Glu-Arg-Asn(SEQ ID NO:10);
Glu-Glu-Ala-Glu-Asn(SEQ ID NO:11); Glu-Glu-Arg-Ala-Arg-Arg(SEQ ID
NO:12); Glu-Glu-Ala-Glu-Pro(SEQ ID NO:13); Glu-Glu-Gly-Glu-Pro(SEQ
ID NO:14); Glu-Glu-Ala-Glu-Cys(SEQ ID NO:15) and
Glu-Glu-Ile-Glu-Gly-Arg(SEQ ID NO:16).
[0030] According to a further aspect the present invention is
related to a polypeptide precursor for a desired polypeptide said
polypeptide precursor having the formula
N-terminal extension-Y.sub.1-*polypeptide*
[0031] wherein Y.sub.1 is Met, Asn, Pro, Gln, Cys or Arg-Arg; the
N-terminal extension has 1-14 amino acid residues as described
above and *polypeptide* is the remaining part of the desired
polypeptide.
[0032] According to a still further aspect the present invention is
related to a polypeptide precursor for a desired polypeptide said
polypeptide precursor having the formula
N-terminal extension-Y.sub.1--Y.sub.2- Y.sub.3-*polypeptide*
[0033] wherein Y.sub.1 is Met, Asn, Pro, Gln, Cys or Arg-Arg;
Y.sub.2 is His or Tyr, Y.sub.3 is Ala, Ser or Gly, the N-terminal
extension has 1-14 amino acid residues as described above and
*polypeptide* is the remaining part of desired polypeptide.
[0034] More specifically, Y.sub.2 is N-terminal amino acid residue
in the desired polypeptide and Y.sub.3 is the second amino acid
residue from the N-terminal end of the desired polypeptide
[0035] According to further aspects the present invention is
related to polynucleotides encoding the claimed polypeptide
precursors and vectors and transformed host cells containing such
polynucleotides.
[0036] Introduction of lipophilic acyl groups in naturally
occurring peptides or analogues thereof has shown to lead to
acylated peptides which have a protracted profile relative to the
native peptide or unmodified analogues. This phenomenon is
disclosed and demonstrated in WO 98/08871 which discloses acylation
of GLP-1 and analogues thereof and in WO 98/08872 which discloses
acylation of GLP-2 and analogues thereof. The lipophilic group may
be introduced by means of mono- or dipeptide spacers as disclosed
in WO 98/08871. Alternatively, the lipophilic group may be
introduced by means of .alpha.-amino-.alpha.,.omega.-dicarboxylic
acid groups as disclosed in WO 00/55119.
[0037] The present polypeptide precursor will contain at least one
lysine group with a free .epsilon.-amino group to be acylated. When
acylating one or more free amino acid groups in a polypeptide
acylation of the free amino group in the N-terminal amino acid
residue is more or less avoidable. Certain methods have been
developed to avoid acylation at the N-terminal amino acid residue,
vide U.S. Pat. No. 5,905,140. The present invention offers an
alternative solution to the problem, i.e. to express an
N-terminally extended precursor of the desired polypeptide. So even
if acylation takes place both at the desired position in the
desired polypeptide and at the N-terminal amino acid residue,
subsequent cleavage of the N-terminal extension from the desired
polypeptide will removed the unwanted acylated amino acid residue.
The precursor molecule can thus be preferentially acylated in the
desired lysine residue which in the case of GLP-1 is the lysine in
position 26. After acylation the acylated precursor molecule is
cleaved by suitable chemical or enzymatic means as described above
and the desired acylated polypeptide can be isolated.
[0038] The acylation step (iii) may be conducted at a pH between 7
and 12. In certain embodiments, the pH will be between 8 and 11.5
or between 9.0 and 10.5 and a pH value of about 9.5 to 10.5 has
proven to be efficient. The temperature will be between minus 5 and
35.degree. C. and will typically be between 0 and 20.degree. C. or
between 15 and 30.degree. C.
DEFINITIONS
[0039] The term "preferential acylating" is meant to include and
acylation process where acylation takes place at one or more
preferred positions in the molecule in a higher degree than at
other positions in the same molecule. Thus, the acylation at the
preferred positions is preferably at least 50, more preferred at
least 80 and most preferred 90-100% of the total acylation.
[0040] With "N-terminal extension" is meant a polypeptide sequence
removably attached to the N-terminal amino acid residue in the
desired polypeptide. The N-terminal extension may be 1-15 amino
acid residues in length and will not comprise a Lys residue. The
N-terminal extension will protect the expressed fusion polypeptide
against proteolytic degradation within the host cell as described
above.
[0041] With "desired polypeptide" is meant the ultimate polypeptide
obtained after cleavage of the N-terminal extension from the
precursor molecule. This expression will cover both the acylated
and non-acylated version of said polypeptide. The "N-terminal
extension" includes the cleavage site for cleavage of the
N-terminal extension from the desired polypeptide's N-terminal end.
It will be understood that whenever a specific N-terminal extension
or a sequence being comprised in the N-terminal extension is shown,
then the given C-terminal amino acid residue will be the cleavage
site directly linked to the N-terminal amino acid residue of the
desired polypeptide.
[0042] An example of a desired polypeptide is GLP-1. The amino acid
sequence of GLP-1 is given i.a. by Schmidt et al. (Diabetologia 28
704-707 (1985). Although the interesting pharmacological properties
of GLP-1 (7-37) and analogues thereof have attracted much attention
in recent years only little is known about the structure of these
molecules. The secondary structure of GLP-1 in micelles has been
described by Thorton et al. (Biochemistry 33 3532-3539 (1994)), but
in normal solution, GLP-1 is considered a very flexible
molecule.
[0043] A simple system is used to describe fragments and analogues
of this peptide. Thus, for example, Gly.sup.8GLP-1.sub.(7-37)
designates a fragment of GLP-1 derived from GLP-1.sub.(1-37) by
deleting the amino acid residues Nos. 1 to 6 and substituting the
naturally occurring amino acid residue in position 8 (Ala) by Gly.
Similarly,
Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1.sub.(7-37)
designates GLP-1.sub.(7-37) wherein the .epsilon.-amino group of
the Lys residue in position 26 has been tetradecanoylated.
[0044] Other examples of a desired polypeptides are GLP-2 and
glucagon both belonging to the GRF (growth hormone releasing
factor) family of peptides having a His or Tyr in the N-terminal
position and Ser, Ala or Gly in the next position, vide Adelhorts
K. et al., The Journal of Biological Chemistry (1994) p
6275-6278).
[0045] "POT" is the Schizosaccharomyces pombe triose phosphate
isomerase gene, and "TPI1" is the S. cerevisiae triose phosphate
isomerase gene.
[0046] With "fibrillation" is meant a process where so called
"fibrils" are formed. "Fibrils" is a well recognized and described
phenomenon and may be composed of antiparallel .beta.-sheets.
Molecules like GLP's with little .alpha.-helical structure and a
very flexible and little tertiary structure are very prone to
aggregation that leads to precipitation and loss of yield if very
crude chemical conditions are not taken in use such as alkaline
treatment at pH.about.12.
[0047] By a "leader" is meant an amino acid sequence consisting of
a pre-peptide (the signal peptide) and a pro-peptide.
[0048] The term "signal peptide" is understood to mean a
pre-peptide which is present as an N-terminal sequence on the
precursor form of a protein. The function of the signal peptide is
to allow the heterologous protein to facilitate translocation into
the endoplasmic reticulum. The signal peptide is normally cleaved
off in the course of this process. The signal peptide may be
heterologous or homologous to the yeast organism producing the
protein. A number of signal peptides may be used with the DNA
construct of the invention including the YPS1 signal peptide
(formally called the YAP3 signal peptide) or any functional
analogue thereof (Egel-Mitani et al. (1990) YEAST 6:127-137 and
U.S. Pat. No. 5,726,038) and the .alpha.-factor signal of the
MF.alpha.1 gene (Thorner (1981) in The Molecular Biology of the
Yeast Saccharomyces cerevisiae, Strathern et al., eds., pp 143-180,
Cold Spring Harbor Laboratory, New York and U.S. Pat. No.
4,870,008.
[0049] The term "pro-peptide" means a polypeptide sequence whose
function is to allow the expressed polypeptide to be directed from
the endoplasmic reticulum to the Golgi apparatus and further to a
secretory vesicle for secretion into the culture medium (i.e.
exportation of the polypeptide across the cell wall or at least
through the cellular membrane into the periplasmic space of the
yeast cell). The pro-peptide may be the yeast .alpha.-factor
pro-peptide, vide U.S. Pat. Nos. 4,546,082 and 4,870,008.
Alternatively, the pro-peptide may be a synthetic pro-peptide,
which is to say a pro-peptide not found in nature. Suitable
synthetic pro-peptides are those disclosed in U.S. Pat. Nos.
5,395,922; 5,795,746; 5,162,498 and WO 98/32867. The pro- peptide
will preferably contain an endopeptidase processing site at the
C-terminal end, such as a Lys-Arg sequence or any functional analog
thereof.
[0050] The polynucleotide sequence of the invention may be prepared
synthetically by established standard methods, e.g. the
phosphoamidite method described by Beaucage et al. (1981)
Tetrahedron Letters 22:1859-1869, or the method described by
Matthes et al. (1984) EMBO Journal 3:801-805. According to the
phosphoamidite method, oligonucleotides are synthesized, for
example, in an automatic DNA synthesizer, purified, duplexed and
ligated to form the synthetic DNA construct. A currently preferred
way of preparing the DNA construct is by polymerase chain reaction
(PCR).
[0051] The polynucleotide sequence of the invention may also be of
mixed genomic, cDNA, and synthetic origin. For example, a genomic
or cDNA sequence encoding a leader peptide may be joined to a
genomic or cDNA sequence encoding the precursor molecule of the
invention, after which the DNA sequence may be modified at a site
by inserting synthetic oligonucleotides encoding the desired amino
acid sequence for homologous recombination in accordance with
well-known procedures or preferably generating the desired sequence
by PCR using suitable oligonucleotides.
[0052] The invention encompasses a vector which is capable of
replicating in the selected microorganism or host cell and which
carries a polynucleotide sequence encoding the precursor molecule
of the invention. The recombinant vector may be an autonomously
replicating vector, i.e., a vector which exists as an
extra-chromosomal entity, the replication of which is independent
of chromosomal replication, e.g., a plasmid, an extra-chromosomal
element, a mini-chromosome, or an artificial chromosome. The vector
may contain any means for assuring self-replication. Alternatively,
the vector may be one which, when introduced into the host cell, is
integrated into the genome and replicated together with the
chromosome(s) into which it has been integrated. Furthermore, a
single vector or plasmid or two or more vectors or plasmids which
together contain the total DNA to be introduced into the genome of
the host cell, or a transposon may be used. The vector may be
linear or closed circular plasmids and will preferably contain an
element(s) that permits stable integration of the vector into the
host cell's genome or autonomous replication of the vector in the
cell independent of the genome.
[0053] In a preferred embodiment, the recombinant expression vector
is capable of replicating in yeast. Examples of sequences which
enable the vector to replicate in yeast are the yeast plasmid 2
.mu.m replication genes REP 1-3 and origin of replication.
[0054] The vectors of the present invention preferably contain one
or more selectable markers which permit easy selection of
transformed cells. A selectable marker is a gene the product of
which provides for biocide or viral resistance, resistance to heavy
metals, prototrophy to auxotrophs, and the like. Examples of
bacterial selectable markers are the dal genes from Bacillus
subtilis or Bacillus licheniformis, or markers which confer
antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol or tetracycline resistance. Selectable markers for
use in a filamentous fungal host cell include amdS (acetamidase),
argB (ornithine carbamoyl-transferase), pyrG
(orotidine-5'-phosphate decarboxylase) and trpC (anthranilate
synthase. Suitable markers for yeast host cells are ADE2, HIS3,
LEU2, LYS2, MET3, TRP1, and URA3. A preferred selectable marker for
yeast is the Schizosaccharomyces pompe TPI gene (Russell (1985)
Gene 40:125-130).
[0055] In the vector, the polynucleotide sequence is operably
connected to a suitable promoter sequence. The promoter may be any
nucleic acid sequence which shows transcriptional activity in the
host cell of choice including mutant, truncated, and hybrid
promoters, and may be obtained from genes encoding extra-cellular
or intra-cellular polypeptides either homologous or heterologous to
the host cell.
[0056] Examples of suitable promoters for directing the
transcription in a bacterial host cell, are the promoters obtained
from the E. coli lac operon, Streptomyces coelicolor agarase gene
(dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus
licheniformis alpha-amylase gene (amyL), Bacillus
stearothermophilus maltogenic amylase gene (amyM), Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), and Bacillus
licheniformis penicillinase gene (penP). Examples of suitable
promoters for directing the transcription in a filamentous fungal
host cell are promoters obtained from the genes for Aspergillus
oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase,
Aspergillus niger neutral alpha-amylase, and Aspergillus niger acid
stable alpha-amylase. In a yeast host, useful promoters are the
Saccharomyces cerevisiae MF.alpha.1, TPI, ADHm Gal or PGK
promoters.
[0057] The polynucleotide construct of the invention will also
typically be operably connected to a suitable terminator. In yeast
a suitable terminator is the TPI terminator (Alber et al. (1982) J.
Mol. Appl. Genet. 1:419-434) or the CYC1 terminator.
[0058] The procedures used to ligate the polynucleotide sequence of
the invention, the promoter and the terminator, respectively, and
to insert them into a suitable vector containing the information
necessary for replication in the selected host, are well known to
persons skilled in the art. It will be understood that the vector
may be constructed either by first preparing a DNA construct
containing the entire DNA sequence encoding the precursor molecule
of the invention, and subsequently inserting this fragment into a
suitable expression vector, or by sequentially inserting DNA
fragments containing genetic information for the individual
elements followed by ligation.
[0059] The present invention also relates to recombinant host
cells, comprising a polynucleotide sequence encoding the precursor
molecule of the invention. A vector comprising such polynucleotide
sequence is introduced into the host cell so that the vector is
maintained as a chromosomal integrant or as a self-replicating
extra-chromosomal vector as described earlier. The term "host cell"
encompasses any progeny of a parent cell that is not identical to
the parent cell due to mutations that occur during replication. The
host cell may be a prokaryote or a eukaryote cell. Useful
prokaryotes are bacterial cells such as gram positive bacteria
including Bacillus and Streptomyces cells, or gram negative
bacteria such as E. coli and Pseudomonas sp. Cells. Eukaryote cells
may be mammalian, insect, plant, or fungal cells. In a one
embodiment, the host cell is a yeast cell. The yeast organism used
in the process of the invention may be any suitable yeast organism
which, on cultivation, produces large amounts of the precursor
molecule. Examples of suitable yeast organisms are strains selected
from the yeast species Saccharomyces cerevisiae, Saccharomyces
kluyveri, Schizosaccharomyces pombe, Sacchoromyces uvarum,
Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia
methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp.,
Candida utilis, Candida cacaoi, Geotrichum sp., and Geotrichum
fermentans.
[0060] The transformation of the yeast cells may for instance be
effected by protoplast formation followed by transformation in a
manner known per se. The medium used to cultivate the cells may be
any conventional medium suitable for growing yeast organisms. The
secreted precursor of the invention may then be recovered from the
medium by conventional procedures including separating the yeast
cells from the medium by centrifugation, filtration or catching the
precursor by an ion exchange matrix or by a reverse phase
absorption matrix, precipitating the proteinaceous components of
the supernatant or filtrate by means of a salt, e.g. ammonium
sulphate, followed by purification by a variety of chromatographic
procedures, e.g. ion exchange chromatography, affinity
chromatography, or the like.
[0061] In the present text, the designation "an analogue" is used
to designate a peptide wherein one or more amino acid residues of
the parent peptide have been substituted by another amino acid
residue and/or wherein one or more amino acid residues of the
parent peptide have been deleted and/or wherein one or more amino
acid residues have been added to the parent peptide. Such addition
can take place either at the N-terminal end or at the C-terminal
end of the parent peptide or both.
[0062] The term "derivative" is used in the present text to
designate a peptide in which one or more of the amino acid residues
of the parent peptide have been chemically modified, e.g. by
alkylation, acylation, ester formation or amide formation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] FIG. 1 shows the plasmid pKV304 which contain DNA encoding
Arg.sup.34GLP-1.sub.(1-37) under regulatory control of the TPI
promoter and -terminator and the MFalpha prepro sequence. This
plasmid is the starting plasmid for making expression plasmids for
the precursor molecules according to the present invention.
EXAMPLES
Example 1
Expression of N-terminally Extended Arg.sup.34GLP-1.sub.(7-37)
[0064] The host strain ME1719 is a diploid strain and has a
phenotype which lacks two aspartyl protease activities, i.e. YPS1
(previously called YAP3) which cleaves C-terminal side of mono- or
dibasic amino acid residues (Egel-Mitani, et al., YEAST 6: 127-137,
1990) and PEP4 a vacuolar protease A responsible for activation of
other proteases such as protease B, carboxypeptidase Y,
aminopeptidase I, RNase, alkaline phosphatase, acid threhalase and
exopolyphosphatase. Moreover the triose phosphate isomerase gene
(TPI) has been disrupted which phenotype makes it possible to
utilize glucose in transformants grown on glucose containing
medium. The genetic background of ME1719 is MATa/.alpha.
.DELTA.yps1::ura3/.DELTA.yps1::URA3 pep4-3/pep4-3
.DELTA.tpi::LEU2/.DELTA- .tpi::LEU2 leu2/leu2 .DELTA.ura3/
.DELTA.ura3.
[0065] Expression plasmids containing the N-terminally extended
Arg.sup.34GLP-1.sub.(7-37) were made as follows: Plasmid pKV304
containing DNA encoding Arg.sup.34GLP-1.sub.(7-37) without an
N-terminal extension was digested with either Eagl+Ncol or
Eagl+Asp718. After agarose electrophoresis and GeneClean.TM. III
purification, fragments of 1.4 kb and 10 kb, respectively were
isolated. Oligonucleotide adaptors corresponding to various
N-terminal extensions of Arg.sup.34GLP-1.sub.(7-- 37) containing
NcoI and Asp718 cleavage sites were likewise purified as described
above. The 1.4 kb fragment (Eagl+NcoI), 10 kb fragment
(Eagl+Asp7l8) and the adaptor fragment designed for the N-terminal
extension of Arg.sup.34GLP-1.sub.(7-37) (NcoI+Asp718) were ligated
and transformed in E. coli strain MT172 and plasmid DNA was
sequenced to verify the correct N-terminally extended
Arg.sup.34GLP-1.sub.(7-37).
[0066] Plasmid DNA was then transformed into yeast strain ME1719
and yeast transformants were isolated twice on MUPD selective
plates. Yeast cells were cultured in 5 ml MUPD medium for 3 days at
30.degree. C. and culture supernatants were analyzed by HPLC and
MALDI-MS (Matrix Assisted Laser Desorption/Inonisation Mass
Spectrometry).
[0067] Table 1 shows the different GLP-1 precursors and the yield
compared to a control with no N-terminal extension.
1TABLE 1 Yield Extension Polypeptide Percentage of control None
(control) Arg.sup.34GLP-1.sub.(7-37) 100 EEM
Arg.sup.34GLP-1.sub.(7-37) 199 EEAEM, SEQ ID NO: 1
Arg.sup.34GLP-1.sub.(7-37) 247 EEAEN, SEQ ID NO: 2
Arg.sup.34GLP-1.sub.(7-37) 249 EEAERR, SEQ ID NO: 3
Arg.sup.34GLP-1.sub.(7-37) 159 EERARR, SEQ ID NO: 12
Arg.sup.34GLP-1.sub.(7-37) 40 Q Arg.sup.34GLP-1.sub.(7-37) 107 EPQ
Arg.sup.34GLP-1.sub.(7-37) 232 EAQ Arg.sup.34GLP-1.sub.(7-3- 7) 146
EAEAQ, SEQ ID NO: 5 Arg.sup.34GLP-1.sub.(7-37) 227 EAEAEAQ, SEQ ID
NO: 6 Arg.sup.34GLP-1.sub.(7-37) 251 EEAEP, SEQ ID NO: 13
Arg.sup.34GLP-1.sub.(7-37) 229 EEGEP, SEQ ID NO: 14
Arg.sup.34GLP-1.sub.(7-37) 171 EEGCTSIC, SEQ ID NO: 7
Arg.sup.34GLP-1.sub.(7-37) 68 EHGCTSIC, SEQ ID NO: 8
Arg.sup.34GLP-1.sub.(7-37) 31 EEAEC SEQ, ID NO: 15
Arg.sup.34GLP-1.sub.(7-37) 89, 76* EEIEGR, SEQ ID NO: 16
Arg.sup.34GLP-1.sub.(7-37) 166 *Two peaks
[0068] Acylation of the isolated precursor molecules is conducted
according to the methods described in WO 98/08871, WO 98/09972, WO
00/55119 or U.S. Pat. No. 5,905,140. Cleavage of the N-terminal
extension from the desired molecule will depend on which cleavage
site has been chosen and will then follow standard procedures.
Example 2
Cleavage of Glu-Glu-Ala-Glu-Asn(SEQ ID NO:2)-
Arg.sup.34GLP-1.sub.(7-37) by Hydroxylamine
[0069] Inclusion of asparagine N-terminally to the N-terminal His
residue (His-7) in GLP-1 might open the possibility of cleaving
selectively with hydroxylamine, as no other Asn is present in the
GLP17-37, R34 amino acid sequence. Preferential cleavage at Asn-Gly
motifs is well established in protein technology (Bornstein, P. and
Balian, G. (1970) J. Biol. Chem. 245, 4854-4856 and Blodgett, J.
K., Loudon, G. M., and Collins, K. D. (1985) J. Am. Chem. Soc. 107,
4305-4313). It has been shown that the willingness of Asn-Xxx
cleavage roughly follows the spatial availability, i.e small Xxx
residues are prefered (Geiger, T. and Clarke, S. (1987) J. Biol.
Chem. 262, 785-794). According to the present invention it has
surprisingly been demonstrated that the motive Asn-His constitutes
a cleavage site when only one Asn is present. Potential problems
with the hydroxylamine method might be cleavages at Asp-15 or
Glu-9/Glu-21/Glu-27. However, Asp and Glu residues are typically
much less reactive towards hydroxylamine and imid formation
(compared to Asn). Racemization of His-7 during the process might
be a concern, but given the mild applied conditions, this is most
likely not taking place.
[0070] Glu-Glu-Ala-Glu-Asn(SEQ ID NO:2)-GLP-1 was expressed in
yeast and purified as previously described. Afterwards the peptide
was acylated by use of
N.epsilon.-palmitoyl-Glu-.gamma.-succinimidyl-.alpha.-tert-butyl
ester and deprotected by use of TFA. Reverse-phase HPLC-MS
identified the desired product, Glu-Glu-Ala-Glu-Asn(SEQ ID
NO:2)-NN2211 acylated in position Lys-26 in a yield of 80%. 20% of
the acylation mixture was acylated both in Lys-26 and N-terminally.
Since cleavage of both these peptides at Asn-His should yield the
same product Arg.sup.34GLP-1.sub.(7-- 37) it was decided to do the
exploratory studies with the 80/20 mixture.
[0071] Screening of cleavage conditions was performed as
follows:
2 Time/hours 2 4 20 Temperature/.degree. C. 45 65 85 pH 7 8 9
Conc., NH.sub.2OH/M 0.5 1 3 5
[0072] Optimal conditions in the screened ranges were found to be
20 hours, 45.degree. C., pH 7, 3-5 M hydroxylamine with cleavage
yields of 80-90 % as judged from HPLC. Shorter times or lower
hydroxylamine concentrations gave incomplete cleavages. Higher
temperatures or higher pH's gave increasing levels of
side-products.
Example 3
Cleavage of Non-acylated Glu-Glu-Ile-Glu-Gly-Arg(SEQ ID NO:16)-
Arg.sup.34GLP-1.sub.(7-37) with FXa
[0073] Glu-Glu-Ile-Glu-Gly-Arg(SEQ ID NO:16)-
Arg.sup.34GLP-1.sub.(7-37) was expressed and recovered as
previously described. 1 mg lyophilized product was dissolved in 10
ml 0.02 M Tris and 2 mM CaCl.sub.2, pH 7,5. At time zero 40 units
of FXa (Amersham Pharmacia Biotech, Product number: 27-0849-01) was
added and the incubation was stopped after 2 hours at 30.degree. C.
in a water bath by dilution 1:1 with 1 M HAc. Determination by HPLC
and MALDI-TOF showed 82% correctly cleaved
Arg.sup.34GLP-1.sub.(7-37) and 18% non-cleaved product
Glu-Glu-Ile-Glu-Gly-Arg(SEQ ID NO:16)- Arg.sup.34GLP-1.sub.(7-37).
Arg.sup.34GLP-1.sub.(7-37) can then be acylated by well established
known methods.
Example 4
Cleavage of Non-acylated Glu-Glu-Ala-Glu-Arg-Arg(SEQ ID NO:3)-
Arg.sup.34GLP-1.sub.(7-37) with Kexin
[0074] Glu-Glu-Ala-Glu-Arg-Arg(SEQ ID NO:3)-
Arg.sup.34GLP-1.sub.(7-37) was expressed and recovered as
previously described. 0.37 mg lyophilized product was dissolved in
900 .mu.l 0.1 M NaAc and 5 mM CaCl.sub.2, pH 6.0. At time zero 100
.mu.l of soluble C-terminal truncated kexin was added and the
mixture was incubated in a water bath at 30.degree. C. After 1 hour
the reaction was stopped by addition of 2 M HAc 1:1 and cleavage
was determined by HPLC and MALDI-MS. 66.9% was cleaved to
Arg.sup.34GLP-1.sub.(7-37) and the rest was uncleaved
Glu-Glu-Ala-Glu-Arg-Arg(SEQ ID NO:3)- Arg.sup.34GLP-1.sub.(7-37).
Arg.sup.34GLP-1.sub.(7-37) can then be acylated by well established
known methods.
Example 5
Cleavage of Non-acylated Glu-Glu-Ala-Glu-Pro(SEQ ID NO:13 )-
Arg.sup.34GLP-1.sub.(7-37) with Prolylendopeptidase
[0075] Glu-Glu-Ala-Glu-Pro(SEQ ID NO:13
)-Arg.sup.34GLP-1.sub.(7-37) was expressed and recovered as
previously described. 11.4 mg lyophilized product was dissolved in
50 ml 0.02 M Tris, HCl, pH 7.5. At time zero 500 .mu.l was added of
prolylendopeptidase (Sphingomonas capsulata prolylendopeptidase
expressed in E.coli according to Kanatani et al, Archives of
Biochemistry and Biophysics, 358, 141-148 (1998). Cleavage and
formation of Arg.sup.34GLP-1.sub.(7-37) are detected after 30 min
incubation in a water bath at 30.degree. C.
Arg.sup.34GLP-1.sub.(7-37) can now be acylated by well established,
known methods.
Sequence CWU 1
1
16 1 5 PRT Artificial Sequence Synthetic 1 Glu Glu Ala Glu Met 1 5
2 5 PRT Artificial Sequence Synthetic 2 Glu Glu Ala Glu Asn 1 5 3 6
PRT Artificial Sequence Synthetic 3 Glu Glu Ala Glu Arg Arg 1 5 4 4
PRT Artificial Sequence Synthetic 4 Gln Glu Pro Gln 1 5 5 PRT
Artificial Sequence Synthetic 5 Glu Ala Glu Ala Gln 1 5 6 7 PRT
Artificial Sequence Synthetic 6 Glu Ala Glu Ala Glu Ala Gln 1 5 7 8
PRT Artificial Sequence Synthetic 7 Glu Glu Gly Cys Thr Ser Ile Cys
1 5 8 8 PRT Artificial Sequence Synthetic 8 Glu His Gly Cys Thr Ser
Ile Cys 1 5 9 5 PRT Artificial Sequence Synthetic 9 Glu Glu Ala Arg
Met 1 5 10 4 PRT Artificial Sequence Synthetic 10 Glu Glu Arg Asn 1
11 5 PRT Artificial Sequence Synthetic 11 Glu Glu Ala Glu Asn 1 5
12 6 PRT Artificial Sequence Synthetic 12 Glu Glu Arg Ala Arg Arg 1
5 13 5 PRT Artificial Sequence Synthetic 13 Glu Glu Ala Glu Pro 1 5
14 5 PRT Artificial Sequence Synthetic 14 Glu Glu Gly Glu Pro 1 5
15 5 PRT Artificial Sequence Synthetic 15 Glu Glu Ala Glu Cys 1 5
16 6 PRT Artificial Sequence Synthetic 16 Glu Glu Ile Glu Gly Arg 1
5
* * * * *