U.S. patent application number 12/321006 was filed with the patent office on 2009-09-17 for catalytic domains of beta(1,4)-galactosyl transferase i having altered donor and acceptor specificities, domains that promote in vitro protein folding, and methods for their use.
This patent application is currently assigned to Govt. of the US, as represented by the secretary, Dept. of Health and Human Services. Invention is credited to Pradman Qasba, Boopathy Ramakrishnan.
Application Number | 20090233345 12/321006 |
Document ID | / |
Family ID | 32718075 |
Filed Date | 2009-09-17 |
United States Patent
Application |
20090233345 |
Kind Code |
A1 |
Qasba; Pradman ; et
al. |
September 17, 2009 |
Catalytic domains of beta(1,4)-galactosyl transferase i having
altered donor and acceptor specificities, domains that promote in
vitro protein folding, and methods for their use
Abstract
Disclosed are methods and compositions that can be used to
synthesize oligosaccharides; mutants of galactosyltransferases
having altered donor and acceptor specificity; methods for
increasing the immunogenicity of an antigen; and polypeptide stem
regions that can be used to promote in vitro folding of
polypeptides, such as the catalytic domain from a
galactosyltransferase.
Inventors: |
Qasba; Pradman; (Bethesda,
MD) ; Ramakrishnan; Boopathy; (Frederick,
MD) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
PO BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
Govt. of the US, as represented by
the secretary, Dept. of Health and Human Services
Rockville
MD
|
Family ID: |
32718075 |
Appl. No.: |
12/321006 |
Filed: |
January 13, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11178230 |
Jul 8, 2005 |
7482133 |
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12321006 |
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PCT/US2004/000470 |
Jan 9, 2004 |
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11178230 |
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60439298 |
Jan 10, 2003 |
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60450250 |
Feb 25, 2003 |
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Current U.S.
Class: |
435/193 |
Current CPC
Class: |
C12Y 204/01133 20130101;
C12N 9/1051 20130101 |
Class at
Publication: |
435/193 |
International
Class: |
C12N 9/10 20060101
C12N009/10 |
Goverment Interests
GOVERNMENT FUNDING
[0002] The invention described herein was developed with support
from the National Institutes of Health, under contract
NO1-CO-12400. The U.S. Government may have certain rights in the
invention.
Claims
1.-11. (canceled)
12. An isolated catalytic domain from a
.beta.(1,4)-galactosyltransferase I that can catalyze formation of
a galactose-.beta.(1,4)-N-acetylglucosamine-6-SO.sub.3 bond.
13. The catalytic domain of claim 12, wherein (a) the catalytic
domain has conservative amino acid exchanges at amino acid
positions 279 and 280, or (b) serine is exchanged for lysine at
amino acid position 279, and threonine is exchanged for
phenylalanine at amino acid position 280.
14.-51. (canceled)
52. A kit comprising packaging material, and a
.beta.(1,4)-galactosyltransferase I catalytic domain that: can
catalyze formation of a
galactose-.beta.(1,4)-N-acetylglucosamine-6-SO.sub.3 bond.
53. The kit of claim 52, further comprising a donor.
54. The kit of claim 53, wherein the donor is selected from the
group consisting of UDP-galactose, UDP-mannose,
UDP-N-acetylglucosamine, UDP-glucose, GDP-mannose,
UDP-N-acetylgalactosamine, UDP-glucuronic acid, GDP-Fucose, and
CMP-N-acetylneuraminic acid.
Description
PRIORITY OF INVENTION
[0001] This application is a divisional application of U.S. patent
application Ser. No. 11/178,230, filed Jul. 8, 2005, which is a
continuation under 35 U.S.C. 111(a) of International Application
No. PCT/US2004/000470 filed Jan. 9, 2004, and published in English
as WO 2004/063344 on Jul. 29, 2004, which claims priority from U.S.
Provisional Application No. 60/439,298, filed 10 Jan. 2003, and
U.S. Provisional Application No. 60/450,250, filed 25 Feb. 2003,
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The invention relates generally to
.beta.(1,4)-galactosyltransferase I mutants having altered donor
and acceptor specificities, and methods of use thereof. In
addition, the invention relates to methods for using the
.beta.(1,4)-galactosyltransferase I mutants to increase the
immunogenicity of an antigen, such as vaccines, and for
synthesizing saccharide compositions.
BACKGROUND OF THE INVENTION
[0004] Oligosaccharides are chains composed of saccharide units,
which are commonly known as sugars. Of the biological polymer
families, oligosaccharides are the least studied, due in part to
the difficulty of sequencing and synthesizing their complex sugar
chain. Currently, no generally applicable synthetic technique for
synthesizing oligosaccharides is available.
[0005] Intensive research efforts have been devoted to
carbohydrates and molecules comprising carbohydrate fragments, such
as glycolipids and glycoproteins. Research interest in these
moieties has been largely due to the recognition that interaction
between proteins and carbohydrates are involved in a wide array of
biological recognition events, including fertilization, molecular
targeting, intracellular recognition, and viral, bacterial, and
fungal pathogenesis. It is now widely appreciated that the
oligosaccharide portions of glycoproteins and glycolipids mediate
cell-cell interactions, cell-ligand interactions,
cell-extracellular matrix interactions, and cell-pathogen
interactions.
[0006] It is thought that many of these interactions can be
inhibited by oligosaccharides that have the same sugar sequence and
stereochemistry found on the active portion of a glycoprotein or
glycolipid involved in the interactions. The oligosaccharides are
believed to compete with the glycoproteins and glycolipids for
binding sites on the receptor proteins. For example, the
disaccharide galactosyl-.beta.(1,4)-N-acetylglucosamine is believed
to be one component of the glycoprotein which interacts with
receptors in the plasma membrane of liver cells. Thus,
oligosaccharides and other saccharide compositions that mimic
ligands recognized and bound by cellular receptors are thought to
be useful in applications that include diagnostics and
therapeutics.
[0007] In addition to mediating numerous cellular interactions,
many oligosaccharides are recognized by the immune system. For
example, Anti-Gal, a naturally occurring antibody present in all
humans, specifically interacts with the carbohydrate epitope
Gal-.alpha.(1-3)Gal-.beta.(1-4)GlcNAc-R (.alpha.-galactosyl
epitope). This antibody does not interact with any other known
carbohydrate epitope produced by mammalian cells (Galili, Springer
Seminar Immunopathology, 15:153 (1993)). Anti-Gal constitutes
approximately 1% of circulating IgG (Galili et al., J. Exp. Med.,
160:1519 (1984)) and is also found in the form of IgA and IgM
(Davine et al., Kidney Int., 31:1132 (1987); Sandrin et al., Proc.
Natl. Acad. Sci., 90:11391 (1993)). It is produced by 1% of
circulating B-lymphocytes (Galili et al., Blood, 82:2485 (1993)).
Accordingly, the ability of carbohydrates to elicit an immune
response can be utilized to increase the effectiveness of vaccines
against many types of pathogens by linking such a carbohydrate to a
vaccine to increase the immune response to the vaccine.
[0008] There has been relatively little effort to test
oligosaccharides as therapeutic agents for humans or animal
diseases however, as methods to synthesize oligosaccharides have
been unavailable as noted above. Limited types of small
oligosaccharides can be custom-synthesized by organic chemical
methods, but the cost of such compounds is typically prohibitively
high. In addition, it is very difficult to synthesize
oligosaccharides stereospecifically and the addition of some
sugars, such as sialic acid and fucose, has not been effectively
accomplished because of the extreme lability of their bonds.
Improved, generally applicable methods for oligosaccharide
synthesis are thereby desired for the production of large amounts
of widely varying oligosaccharides for therapeutic purposes.
Accordingly, the present invention provides enzymes and methods
that can be used to promote the chemical linkage of numerous sugars
that have previously been difficult to link.
SUMMARY OF THE INVENTION
[0009] The invention provides altered
.beta.(1,4)-galactosyltransferase I catalytic domains that catalyze
the formation of a glucose-.beta.(1,4)-N-acetylglucosamine bond at
a greater rate than the catalytic domain of the corresponding
wild-type enzyme. The invention also provides
.beta.(1,4)-galactosyltransferase I catalytic domains that catalyze
formation of N-acetylgalactosamine-.beta.(1,4)-N-acetylglucosamine
bonds; N-acetylgalactosamine-.beta.(1,4)-glucose bonds;
N-acetylglucosamine-.beta.(1,4)-N-acetylglucosamine bonds;
mannose-.beta.(1,4)-N-acetylglucosamine bonds; and
galactose-.beta.(1,4)-N-acetylglucosamine-6-SO.sub.3 bonds. The
invention also provides polypeptides that contain each of the
aforementioned catalytic domains.
[0010] Also provided by the invention are amino acid segments that
promote in vitro folding of catalytic domains of
galactosyltransferases, such as .beta.(1,4)-galactosyltransferase
I, and mutants of galactosyltransferases.
[0011] The invention provides nucleic acid segments that encode the
aforementioned .beta.(1,4)-galactosyltransferase I catalytic
domains. Expression cassettes and cells that include nucleic acid
segments that encode the aforementioned
.beta.(1,4)-galactosyltransferase I catalytic domains are also
provided.
[0012] Additionally provided are methods to synthesize a
glucose-.beta.(1,4)-N-acetylglucosamine moiety; an
N-acetylgalactosamine-.beta.(1,4)-N-acetylglucosamine moiety; an
N-acetylgalactosamine-.beta.(1,4)-glucose moiety; an
N-acetylglucosamine-.beta.(1,4)-N-acetylglucosamine moiety; a
mannose-.beta.(1,4)-N-acetylglucosamine moiety; and a
galactose-.beta.(1,4)-N-acetylglucosamine-6-SO.sub.3 moiety.
[0013] The invention also provides methods to increase the
immunogenicity of an antigen, and methods to prepare an
oligosaccharide composition, including those having a defined
sequence.
[0014] Further provided by the invention are oligosaccharides
produced through use of the catalytic domains and methods disclosed
herein.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 illustrates the catalytic activity of the wt-Gal-T1
and the mutants Y289L, Y289I, and Y289N. (A) Gal-T activity of
wt-Gal-T1 and of mutants at the saturating concentrations of
.beta.-benzyl-GlcNAc as the acceptor. (B) GalNAc-T activity of the
wt-Gal-T1 and of mutants. The activities are measured with 50 mM
.beta.-benzyl-GlcNAc as acceptors at different concentrations of
the donor. The Gal-T activity of the wt-Gal-T1 was measured with
only 10 mM .beta.-benzyl-GlcNAc because it exhibited inhibition at
50 mM concentration.
[0016] FIG. 2 illustrates the partial .sup.1H NMR spectrum of the
disaccharide (LacdiNAc) product from the GalNAc-T reaction with
GlcNAc as the acceptor. The signal for the GalNAc anomeric proton
is at .delta. 4.58 ppm and for the GlcNAc anomeric proton
corresponding to an .alpha. and .beta. conformer is at .delta. 5.2
and 4.7 ppm. The signals from the acetyl group of each sugar moiety
are at .delta. 2.05 and 2.08 ppm.
[0017] FIG. 3 illustrates the cDNA sequence of the inserts and the
derived protein sequences of the constructs in the pET23a vector
used for studying the effect of the N-terminal stem region (SR) on
the solubility and folding of the catalytic domain (CD) of
.beta.4Gal-T. (A) Sequence of bovine SRCD .beta.4Gal-T1 having Ser
96 in the wild-type of .beta.4Gal-T1 mutated to Ala 96,
corresponding to SEQ ID NO: 9. (B) Sequence of human
SRCD.beta.4Gal-T1, corresponding to SEQ ID NO: 6. The numbers above
the amino acids correspond to the residues that delineate the stem
region. An 11 amino acid extension (not shown) is located at the
amino-terminal end of the protein and is coded by the pET23a vector
leader sequence. The stem region sequence is shaded, followed by
the catalytic domain (CD) sequence. The primers used for PCR
amplification are underlined.
[0018] FIG. 4 shows an SDS-PAGE analysis of the recombinant CD and
SRCD proteins of .beta.b 4Gal-T produced in E. coli. Purified
inclusion bodies were analyzed on 14% SDS-PAGE. Lane 1, bovine
CD.beta.4Gal-T1; Lane 2, bovine SRCD.beta.4Gal-T1; Lane 3, human
CD.beta.4Gal-T1; and Lane 4, human SRCD.beta.4Gal-T1.
[0019] FIG. 5 shows folding of inclusion bodies under different
renaturation conditions: the relative activities of CD.beta.4Gal-T1
and SRCD.beta.4Gal-T1. The relative activity was calculated with
respect to the folding condition I. The largest amount of folded
CD.beta.4Gal-T1 and SRCD.beta.4Gal-T1 was obtained under condition
VIII. The enzyme activity under condition I for CD.beta.4Gal-T1 was
0.052 pmol/min/ng, and for SRCD.beta.4Gal-T1 0.09 pmol/min/ng of
N-acetyllactosamine.
[0020] FIG. 6 shows analysis of the soluble proteins obtained after
renaturation of inclusion bodies by SDS-PAGE electrophoresis. Under
native conditions (-), (without boiling and in the absence of
.beta.-ME) and under denaturing conditions (+) (samples boiled in
the presence of .beta.-ME). Lane 1: bovine CD.beta.4Gal-T1; Lane 2:
bovine SRCD.beta.4Gal-T1; Lane 3: human CD.beta.4Gal-T1; and Lane
4: human SRCD.beta.4Gal-T1. Under native conditions a portion of
the soluble SRCD sample representing misfolded proteins does not
enter the gel (arrow 1 in lanes 2 and 4).
[0021] FIG. 7 illustrates binding and characterization of folded
CD.beta.4Gal-T1 and SRCD.beta.4Gal-T1 on UDP-agarose columns. The
purity of the proteins was judged using SDS-PAGE analysis under
native conditions in the absence of #ME and without boiling the
samples. The protein sample before loading on the UDP-agarose
column (U) and after eluting with 25 mM EDTA and 1 M NaCl (B).
Panel (A) shows the results with the human CD.beta.4Gal-T1 before
and after passing through the UDP-agarose column, (bovine
CD.beta.4Gal-T1 [not shown] behaves in the same way). Panel (B)
shows the results with bovine SRCD.beta.4Gal-T1, and Panel (C)
shows the results with human SRCD.beta.4Gal-T1. The arrows at the
top of the lanes in panels B and C show the presence of soluble but
misfolded proteins.
[0022] FIG. 8 shows the enzymatic activity of the soluble human and
bovine SRCD.beta.4Gal-T1 proteins before and after UDP-agarose
purification. The galactosyltransferase activities were measured as
described herein. Specific activities of these proteins before
binding ((-), black bars) and of the eluates after passing through
the UDP-agarose column ((+), cross-hatched bars) are shown. (A)
Buffer condition I (without PEG-4000 and L-arginine), showed more
misfolded molecules in the human SRCD.beta.4Gal-T1 compared to
bovine SRCD.beta.4Gal-T1. (B) Condition VIII (with PEG-4000 and
L-arginine) showed more properly folded proteins in the human
SRCD.beta.4Gal-T1 compared to bovine SRCD.beta.4Gal-T1 (black
bars). CD.beta.4Gal-T1 showed difference in the amounts of folded
proteins before and after binding on the UDP-agarose columns either
with folding condition I or condition VIII (data not shown).
[0023] FIG. 9 is a schematic diagram of the in vitro folding of CD
and SRCD domains of .beta.4Gal-T1. The unfolded CD and SRCD domains
during the in vitro folding step are thought to generate folding
intermediates (I) and (II), respectively, which produce misfolded
and properly folded molecules. The misfolded molecules of CD, (III)
are mostly insoluble and precipitate out, whereas properly folded
molecules (IV) are soluble, remain in solution and bind to a
UDP-agarose column. SRCD misfolded (III') and properly folded
molecules (IV') are mostly soluble. Due to the solubility effect of
SR domain the proportion of the properly folded SRCD molecules
increased during the folding process. The misfolded SRCD molecules,
although soluble, did not significantly bind to UDP-agarose columns
(III'). Properly folded SRCD molecules bind (IV') and elute from
the UDP-agarose column. The presence of PEG-4000 and L-arginine in
the folding solution increased the proportion of the properly
folded molecules.
DETAILED DESCRIPTION OF THE INVENTION
[0024] .beta.1,4)-galactosyltransferase I catalyzes the transfer of
galactose from the donor, UDP-galactose, to an acceptor,
N-acetylglucosamine, to form a
galactose-.beta.(1,4)-N-acetylglucosamine bond. This reaction
allows galactose to be linked to an N-acetylglucosamine that may
itself be linked to a variety of other molecules. Examples of these
molecules include other sugars and proteins. The reaction can be
used to make many types of molecules having great biological
significance. For example,
galactose-.beta.(1,4)-N-acetylglucosamine linkages are important
for many recognition events that control how cells interact with
each other in the body, and how cells interact with pathogens. In
addition, numerous other linkages of this type are also very
important for cellular recognition and binding events as well as
cellular interactions with pathogens, such as viruses. Therefore,
methods to synthesize these types of bonds have many applications
in research and medicine to develop pharmaceutical agents and
improved vaccines that can be used to treat disease.
[0025] The present invention is based on the surprising discovery
that the enzymatic activity of .beta.(1,4)-galactosyltransferase
can be altered such that the enzyme can make chemical bonds that
are very difficult to make by other methods. These alterations
involve mutating the enzyme such that the mutated enzyme can
transfer many different types of donors, sugars for example, to
many different types of acceptors. Therefore, the mutated
.beta.(1,4)-galactosyltransferases of the invention can be used to
synthesize a variety of products that, until now, have been very
difficult and expensive to produce.
[0026] The invention also provides amino acid segments that promote
the proper folding of a galactosyltransferase catalytic domain. The
amino acid segments may be used to properly fold the
galactosyltransferase catalytic domains of the invention and
thereby increase their activity. The amino acid segments may also
be used to increase the activity of galactosyltransferases that are
produced recombinantly. Accordingly, use of the amino acid segments
according to the invention allows for production of
.beta.(1,4)-galactosyltransferases having increased enzymatic
activity relative to .beta.(1,4)-galactosyltransferases produced in
the absence of the amino acid segments.
DEFINITIONS
[0027] Abbreviations: stem region/catalytic domain
.beta.(1,4)-Galactosyltransferase I (SRCD.beta.4Gal-T1); catalytic
domain of .beta.(1,4)-Galactosyltransferase I (CD.beta.4Gal-T1);
.beta.(1,4)-Galactosyltransferase I (.beta.4Gal-T1); catalytic
domain (CD); stem region (SR); wild-type (wt);
galactosyltransferase activity (Gal-T); beta-mercaptoethanol
(.beta.-ME); N-acetylgalactosamine transferase activity (GalNAc-T);
.alpha.-Lactalbumin (LA).
[0028] The term "acceptor" refers to a molecule or structure onto
which a donor is actively linked through action of a catalytic
domain of a galactosyltransferase, or mutant thereof. Examples of
acceptors include, but are not limited to, carbohydrates,
glycoproteins, and glycolipids.
[0029] The term "catalytic domain" refers to an amino acid segment
which folds into a domain that is able to catalyze the linkage of a
donor to an acceptor. For example, a catalytic domain may be from,
but is not limited to, bovine .beta.(1,4)-Galactosyltransferase I
(Seq ID NO: 5), the catalytic domain from human
.beta.(1,4)-Galactosyltransferase I (Seq ID NO: 3), or the
catalytic domain from mouse .beta.(1,4)-Galactosyltransferase I
(Seq ID NO: 4). A catalytic domain may have an amino acid sequence
found in a wild-type enzyme, or may have an amino acid sequence
that is different from a wild-type sequence. For example, a
catalytic domain may have an amino acid sequence that corresponds
to amino acid residues 130402 of SEQ ID NO: 5, expect that the
lysine is exchanged with arginine at amino acid position 228.
[0030] The term "donor" refers to a molecule that is actively
linked to an acceptor molecule through the action of a catalytic
domain of a galactosyltransferase, or mutant thereof. A donor
molecule can include a sugar, or a sugar derivative. Examples of
donors include, but are not limited to, UDP-galactose, UDP-mannose,
UDP-N-acetylglucosamine, UDP-glucose, GDP-mannose,
UDP-N-acetylgalactosamine, UDP-glucuronic acid, GDP-Fucose, and
CMP-N-acetylneuraminic acid. Donors include sugar derivatives that
include active groups, such as cross-linking agents or labeling
agents. Accordingly, oligosaccharides may be prepared according to
the methods of the invention that include a sugar derivative having
a desired characteristic.
[0031] "Expression cassette" as used herein means a DNA sequence
capable of directing expression of a particular nucleotide sequence
in an appropriate host cell, comprising a promoter operably linked
to the nucleotide sequence of interest that is operably linked to
termination signals. It also typically comprises sequences required
for proper translation of the nucleotide sequence. The expression
cassette may be one that is naturally occurring but has been
obtained in a recombinant form useful for heterologous expression.
The expression of the nucleotide sequence in the expression
cassette may be under the control of a constitutive promoter or of
an inducible promoter that initiates transcription only when the
host cell is exposed to some particular external stimulus. In the
case of a multicellular organism, the promoter can also be specific
to a particular tissue or organ or stage of development.
[0032] The terms oligosaccharide and polysaccharide are used
interchangeably herein. These terms refer to saccharide chains
having two or more linked sugars. Oligosaccharides and
polysaccharides may be homopolymers and heteropolymers having a
random sugar sequence or a preselected sugar sequence.
Additionally, oligosaccharides and polysaccharides may contain
sugars that are normally found in nature, derivatives of sugars,
and mixed polymers thereof.
[0033] "Polypeptides" and "Proteins" are used interchangeably
herein. Polypeptides and proteins can be expressed in vivo through
use of prokaryotic or eukaryotic expression systems. Many such
expressions systems are known in the art and are commercially
available. (Clontech, Palo Alto, Calif.; Stratagene, La Jolla,
Calif.). Examples of such systems include, but are not limited to,
the T7-expression system in prokaryotes and the bacculovirus
expression system in eukaryotes. Polypeptides can also be
synthesized in vitro, e.g., by the solid phase peptide synthetic
method or by in vitro transcription/translation systems. Such
methods are described, for example, in U.S. Pat. Nos. 5,595,887;
5,116,750; 5,168,049 and 5,053,133; Olson et al., Peptides, a 301,
307 (1988). The solid phase peptide synthetic method is an
established and widely used method, which is described in the
following references: Stewart et al., Solid Phase Peptide
Synthesis, W. H. Freeman Co., San Francisco (1969); Merrifield, J.
Am. Chem. Soc., 85 2149 (1963); Meienhofer in "Hormonal Proteins
and Peptides," ed.; C. H. Li, Vol. 2 (Academic Press, 1973), pp.
48-267; Bavaay and Merrifield, "The Peptides," eds. E. Gross and F.
Meienhofer, Vol. 2 (Academic Press, 1980) pp. 3-285; and
Clark-Lewis et al., Meth. Enzymol., 287, 233 (1997). These
polypeptides can be further purified by fractionation on
immunoaffinity or ion-exchange columns; ethanol precipitation;
reverse phase HPLC; chromatography on silica or on an
anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;
ammonium sulfate precipitation; gel filtration using, for example,
Sephadex G-75; or ligand affinity chromatography.
[0034] The polypeptides of the invention include polypeptides
having amino acid exchanges, i.e., variant polypeptides, so long as
the polypeptide variant is biologically active. The variant
polypeptides include the exchange of at least one amino acid
residue in the polypeptide for another amino acid residue,
including exchanges that utilize the D rather than L form, as well
as other well known amino acid analogs, e.g., N-alkyl amino acids,
lactic acid, and the like. These analogs include phosphoserine,
phosphothreonine, phosphotyrosine, hydroxyproline,
gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic
acid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid,
penicillamine, ornithine, citruline, N-methyl-alanine,
para-benzoyl-phenylalanine, phenylglycine, propargylglycine,
sarcosine, N-acetylserine, N-formylmethionine, 3-methylhistidine,
5-hydroxylysine, and other similar amino acids and imino acids and
tert-butylglycine.
[0035] Conservative amino acid exchanges are preferred and include,
for example; aspartic-glutamic as acidic amino acids;
lysine/arginine/histidine as basic amino acids; leucine/isoleucine,
methionine/valine, alanine/valine as hydrophobic amino acids;
serine/glycine/alanine/threonine as hydrophilic amino acids.
Conservative amino acid exchange also includes groupings based on
side chains. Members in each group can be exchanged with another.
For example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine. These may be
exchanged with one another. A group of amino acids having
aliphatic-hydroxyl side chains is serine and threonine. A group of
amino acids having amide-containing side chains is asparagine and
glutamine. A group of amino acids having aromatic side chains is
phenylalanine, tyrosine, and tryptophan. A group of amino acids
having basic side chains is lysine, arginine, and histidine. A
group of amino acids having sulfur-containing side chains is
cysteine and methionine. For example, replacement of a leucine with
an isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid may be accomplished to produce a
variant polypeptide of the invention.
I. .beta.(1.4)-Galactosyltransferase I Catalytic Domains of the
Invention.
[0036] A. Catalytic Domains that Catalyze the Formation of a Bond
Between a Donor and an Acceptor to Form
Glucose-.beta.(1.4)-N-acetylglucosamine Bonds.
[0037] It has been discovered that mutation of the donor binding
site of .beta.(1,4)-galactosyltransferase I can broaden the donor
specificity of the enzyme. More specifically, it has been
determined that substitution of amino acid residues located in the
donor binding site of .beta.(1,4)-galactosyltransferase I to
provide greater flexibility and decreased steric hindrance allow
glucose to be bound and chemically bonded to N-acetylglucosamine.
Such mutations provide for broadened donor binding, such as binding
of glucose, while still preserving interaction with amino acid
residues active during catalytic bond formation between the donor
and the acceptor. Without being bound by any theory, an example of
a catalytic residue thought to be important for catalysis is a
glutamic acid positioned at amino acid position 317 (E317) in the
bovine .beta.(1,4)-galactosyltransferase I. This glutamic acid in
bovine .beta.(1,4)-galactosyltransferase I corresponds to a
glutamic acid residue at amino acid position 313 and at amino acid
position 314 in the human and mouse
.beta.(1,4)-galactosyltransferase I respectively. Accordingly, the
invention provides .beta.(1,4)-galactosyltransferase I mutants
having amino acid substitutions, insertions, and deletions that
provide greater flexibility and decreased steric hindrance in the
donor binding site to allow the mutated
.beta.(1,4)-galactosyltransferase I to catalyze chemical bonding of
the donor to an acceptor, such as N-acetylglucosamine or
glucose.
[0038] In some embodiments, the catalytic domains of the invention
have an arginine exchanged with another amino acid at an amino acid
position corresponding to 228 in the bovine
.beta.(1,4)-galactosyltransferase I (SEQ ID NO: 5). An example of a
specific exchange is R228K. The corresponding arginine in the human
and mouse .beta.(1,4)-galactosyltransferase I is located at amino
acid position 224 and 225 (SEQ ID Nos: 3 and 4 respectively). In
mouse, human, and bovine .beta.(1,4)-galactosyltransferase I, the
arginine is located within the amino acid sequence FNRAKLL (SEQ ID
NO: 1). Accordingly, those of skill in the art can readily
determine an equivalent amino acid in other
.beta.(1,4)-galactosyltransferase I catalytic domains.
[0039] In other embodiments, the catalytic domains of the invention
have an arginine exchanged with another amino acid at an amino acid
position corresponding to 228, and an alanine exchanged with
another amino acid at an amino acid position corresponding to 229
in the bovine .beta.(1,4)-galactosyltransferase I.
[0040] Such catalytic domains are exemplified by a catalytic domain
of bovine .beta.(1,4)-galactosyltransferase I having the arginine
at amino acid position 228 exchanged with lysine (R228K), and the
alanine at amino acid position 229 exchanged with glycine (A229G).
The corresponding alanine in the human and mouse
.beta.(1,4)-galactosyltransferase I is located at amino acid
position 225 and 226 (SEQ ID Nos: 3 and 4 respectively). In mouse,
human, and bovine .beta.(1,4)-galactosyltransferase I, the arginine
is located within the amino acid sequence FNRAKLL (SEQ ID NO: 1).
Accordingly, those of skill in the art can readily determine an
equivalent amino acid in other .beta.(1,4)-galactosyltransferase I
catalytic domains.
[0041] B. Catalytic Domains that Catalyze the Formation of a Bond
Between a Donor and an Acceptor to Form
N-acetylgalactosamine-.beta.(1.4)-N-acetylglucosamine Bonds.
[0042] It was postulated that formation of a hydrogen bond between
N-acetylgalactosamine and an amino acid residue adjoining the donor
binding site in .beta.(1,4)-galactosyltransferase I is responsible
for poor transfer of N-acetylgalactosamine to an acceptor. It was
also postulated that mutation of one or more amino acid residues in
the donor binding site in .beta.(1,4)-galactosyltransferase I to
eliminate hydrogen bond formation with N-acetylgalactosamine allows
the mutated .beta.(1,4)-galactosyltransferase I to transfer
N-acetylgalactosamine from a donor to an acceptor more efficiently.
Therefore, the invention includes mutants of
.beta.(1,4)-galactosyltransferase I in which hydrogen bonds that
reduce transfer of N-acetylgalactosamine to an acceptor, such as
N-acetylglucosamine or glucose, are reduced or absent.
[0043] In some embodiments, the catalytic domains of the invention
have a tyrosine exchanged with another amino acid at an amino acid
position corresponding to 289 in the bovine
.beta.(1,4)-galactosyltransferase I (SEQ ID NO: 5). Examples of
specific exchanges are Y289L, Y289I, and Y289N. The corresponding
tyrosine in the human and mouse .beta.(1,4)-galactosyltransferase I
is located at amino acid position 285 and 286 (SEQ ID Nos: 3 and 4
respectively). In mouse, human, and bovine
.beta.(1,4)-galactosyltransferase I, the tyrosine is located within
the amino acid sequence YVQYFGG (SEQ ID NO: 2). Accordingly, those
of skill in the art can readily determine equivalent amino acids in
other .beta.(1,4)-galactosyltransferase I catalytic domains.
[0044] Mutants in which the tyrosine corresponding to that located
at amino acid position 289 in the bovine
.beta.(1,4)-galactosyltransferase I has been exchanged by another
amino acid may optionally include a second mutation corresponding
to amino acid position 342. Such a mutation may include exchange of
cysteine at amino acid position 342 with threonine (C342T).
However, other amino acids may be exchanged for cysteine that
provide and active catalytic domain.
[0045] C. Catalytic Domains that Catalyze the Formation of a Bond
Between a Donor and an Acceptor to Form
N-acetylgalactosamine-.beta.(1.4)-glucose Bonds.
[0046] .beta.(1,4)-galactosyltransferase I mutants, as described
herein, that are able to catalyze chemical bond formation of
N-acetylgalactosamine to an acceptor may be used in conjunction
with .alpha.-lactalbumin to catalyze the formation of
N-acetylgalactosamine-.beta.(1,4)-glucose bonds.
[0047] .alpha.-Lactalbumin is a mammary gland-specific
calcium-binding protein that alters the sugar acceptor specificity
of .beta.(1,4)-galactosyltransferase I toward glucose.
Consequently, .alpha.-lactalbumin may be used to alter the acceptor
specificity .beta.(1,4)-galactosyltransferase I, and mutants
thereof that are described herein, to efficiently catalyze
N-acetylgalactosamine-.beta.(1,4)-glucose bond formation.
Conditions for use of .alpha.-lactalbumin in conjunction with a
galactosyltransferase, or active domain thereof, have been
described (Ramakrishnan et al., J. Biol. Chem., 276:37665
(2001)).
[0048] D. Catalytic Domains that Catalyze the Formation of a Bond
Between a Donor and an Acceptor to form
N-acetylglucosamine-.beta.(1.4)-N-acetylglucosamine Bonds, Oligo
N-acetylgalactosamine-.beta.(1,4)-N-acetylglucosamine, and
mannose-.beta.(1,4) -N-acetylglucosamine.
[0049] The mutations described herein may be combined to create
catalytic domains having selectively altered donor specificities.
For example, a catalytic domain obtained from bovine
.beta.(1,4)-galactosyltransferase I having exchanges at amino acid
positions 228 (R228K) and 289 (Y289L) was able to catalyze the
linkage of N-acetylglucosamine to N-acetylglucosamine to form a
N-acetylglucosamine-.beta.(1,4)-N-acetylglucosamine bond. The same
mutant catalytic domain was able to catalyze the linkage of
N-acetylgalactosamine to N-acetylglucosamine to form oligo
N-acetylgalactosamine-.beta.(1,4)-N-acetylglucosamine. The
broadened donor specificity was further demonstrated by the ability
of the mutant catalytic domain to catalyze the linkage of mannose
to N-acetylglucosamine to form
mannose-.beta.(1,4)-N-acetylglucosamine. Accordingly, numerous
mutant catalytic domains having altered donor specificity may be
created by mutating amino acids corresponding to those at positions
corresponding to 228 and 289 of the bovine
.beta.(1,4)-galactosyltransferase I. As described above, these
amino acid positions may be readily determined in
galactosyltransferase enzymes obtained from other organisms, such
as humans, and mutated in produce additional catalytic domains
having altered donor specificity.
[0050] E. Catalytic Domains that Catalyze the Formation of a Bond
Between a Donor and an Acceptor Having a Bulky Side-Group to Form
for Example, Galactose-.beta.(1.4)-N-acetylglucosamine-6-SO.sub.3
Bonds.
[0051] The acceptor specificity of a catalytic domain obtained from
a galactosyltransferase may be altered to create catalytic domains
capable of transferring a donor onto an acceptor having a bulky
and/or charged side-group.
[0052] An example of such an altered catalytic domain obtained from
bovine .beta.(1,4)-galactosyltransferase I has substitutions at
amino acid positions 279 (K279S) and 280 (F280T). This altered
catalytic domain is able to catalyze the transfer of galactose to
N-acetylglucosamine-6-SO.sub.3 to form
galactose-.beta.(1,4)-N-acetylglucosamine-6-SO.sub.3. Additional
catalytic domains may be created by altering one or more amino acid
residues at positions corresponding to 279 and 280 of the bovine
.beta.(1,4)-galactosyltransferase I. As described above, these
amino acid positions may be readily determined in
galactosyltransferase enzymes obtained from other organisms, such
as humans, and mutated in produce additional catalytic domains
having altered acceptor specificity.
[0053] The amino acids at positions corresponding to 279 and 280 in
the bovine .beta.(1,4)-galactosyltransferase I may be exchanged
individually or together to create many different catalytic domains
having altered acceptor sites able to accept numerous acceptors
having bulky (sterically large) or charged side-groups. Such
altered catalytic domains may be used to catalyze linkage of sugars
from a donor to an acceptor having a desired side-chain.
II. Catalytic Domains of the Invention May be Included within
Full-Length .beta.(1.4)-galactosyltransferase I Enzymes, or in
Recombinant Molecules Containing the Catalytic Domains.
[0054] Peptides of the invention include isolated catalytic
domains, full-length .beta.(1,4)-galactosyltransferase I enzymes
containing a catalytic domain of the invention, as well as
recombinant polypeptides comprising a catalytic domain of the
invention that are linked to additional amino acids. Such
polypeptides may be expressed from DNA constructs and expression
cassettes that are produced through use of recombinant methods.
Such methods have been described. Sambrook et al., Molecular
Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor
Press, Cold Spring Harbor, N.Y. (2001).
[0055] Galactosyltransferase enzymes containing a catalytic domain
of the invention may be produced in soluble form. Methods that may
be used to produce such soluble enzymes have been described (U.S.
Pat. No. 5,032,519). Briefly, a hydrophobic transmembrane anchor
region of a galactosyltransferase is removed to produce an enzyme
that is in soluble form.
[0056] Alternatively, .beta.(1,4)-galactosyltransferase enzymes
containing a catalytic domain of the invention may be produced such
that they are anchored in the membrane of a cell that expresses the
galactosyltransferase. Such enzymes may be produced that are
anchored in the membranes of prokaryotic and eukaryotic cells.
Methods to produce such enzymes have been described (U.S. Pat. No.
6,284,493).
[0057] Briefly, in the case of procaryotes, the signal and
transmembrane sequences of the galactosyltransferase are replaced
by a bacterial signal sequence, capable of effecting localization
of the fusion protein to the outer membrane. Suitable signal
sequences include, but are not limited to those from the major E.
coli lipoprotein Lpp and lam B. In addition, membrane spanning
regions from Omp A, Omp C, Omp F or Pho E can be used in a
tripartite fusion protein to direct proper insertion of the fusion
protein into the outer membrane. Any procaryotic cells can be used
in accordance with the present invention including but not limited
to E. coli, Bacillus sp., and Pseudomonas sp. as representative
examples.
[0058] In another embodiment, the native transmembrane domain of
the galactosyltransferase is replaced by the transmembrane domain
of a bacterial outer membrane protein. In this embodiment, the
galactosyltransferase signal sequence and the bacterial
transmembrane region act in concert to anchor the
galactosyltransferase to the bacterial outer cell membrane. Nearly
any outer membrane bound protein is suitable for this use including
but not limited to Omp A, Omp C, and Omp F, Lpp, and Lam B. The
catalytic portion of the galactosyltransferase should be fused to
an extracellular loop in the bacterial transmembrane region in
order to insure proper orientation of the fusion protein on the
outer membrane surface and not in the cytoplasm or periplasm of the
cell. Insertion of a protein into such a loop region has been
previously reported (Charbit et al., J. Bacteriology, 173:262
(1991); Francisco et al., Proc. Natl. Acad. Sci., 89:2713
(1992)).
[0059] The present invention is also applicable for use with
eukaryotic cells resulting in cell surface expression of
galactosyltransferases in known culturable eucaryotic cells
including but not limited to yeast cells, insect cells, chinese
hamster ovary cells (CHO cells), mouse L cells, mouse A9 cells,
baby hamster kidney cells, C127 cells, COS cells, Sf9 cells, and
PC8 cells.
[0060] In another embodiment of the present invention, the
transmembrane domain of the galactosyltransferase is replaced by
the transmembrane domain of a plasma membrane protein. The
transmembrane domain of any resident plasma membrane protein will
be appropriate for this purpose. The transmembrane portions of the
M6 P/IGF-II receptor, LDL receptor or the transferrin receptor are
representative examples.
[0061] In another embodiment the Golgi retention signal of the
galactosyltransferase is disrupted by site-directed mutagenesis.
This approach mutates the amino acids responsible for localizing
the galactosyltransferase to the Golgi compartment. The resultant
galactosyltransferase is transported to the plasma membrane where
it becomes anchored via its modified transmembrane sequences.
Substitution of isoleucine residues for the native amino acids in
the transmembrane region of the .beta.(1,4)galactosyltransferase
has been shown to preferentially localize the enzyme to the plasma
membrane instead of the Golgi apparatus (Masibay et al., J. Biol.
Chem., 268:9908 (1993)).
III. A Stem Region that Promotes the In Vitro Folding of a
Catalytic Domain of a Galactosyltransferase.
[0062] .beta.(1,4)-galactosyltransferase I is a type II Golgi
resident protein with a short cytoplasmic tail, a transmembrane
domain followed by a stem region and has a globular catalytic
domain that faces the Golgi lumen. When the catalytic domain of
.beta.(1,4)-galactosyltransferase I is expressed in E. coli, it
forms insoluble inclusion bodies. These inclusion bodies can be
collected and then solubilized and folded in vitro to produce
catalytically active domains. Thus, the in vitro folding efficiency
is directly related to the quantity of active enzyme that is
produced from the isolated inclusion bodies. Accordingly, methods
to increase the in vitro folding efficiency would provide increased
production of catalytic domains that can be used to create useful
products.
[0063] The invention provides materials and methods that improve in
vitro folding of catalytic domains from galactosyltransferases that
are related to the use of a stem region (for example, SEQ ID NOs: 6
and 7) of .beta.(1,4)-galactosyltransferase I. It has been
determined that fusion of a stem region from a
.beta.(1,4)-galactosyltransferase I to the amino-terminus of the
catalytic domain of a .beta.(1,4)-galactosyltransferase I produces
increased in vitro folding efficiency of the catalytic domain. This
increase in folding is thought to be universal among
.beta.(1,4)-galactosyltransferase I enzymes and was demonstrated
with both the bovine and human enzymes.
[0064] It has been further discovered that inclusion of PEG-4000
and L-Arg in the folding reaction results in a four-fold to
seven-fold increase in catalytic domains that are natively folded
when compared to refolding of the catalytic domain alone in the
absence of PEG-4000 and L-Arg. PEG-4000 and L-arginine are thought
to beneficially affect the solubility of folding intermediates of
both catalytic domain-proteins (CD-proteins) and stem
region/catalytic domain proteins (SRCD-proteins) during in vitro
folding or protein obtained from inclusion bodies. The presence of
PEG-4000 and L-arginine during in vitro folding enhanced the
formation of both native and misfolded molecules. The processes
involved are schematically shown in FIG. 9. In the case of
CD-proteins, the majority of misfolded proteins are insoluble in
the absence of PEG-4000 and L-arginine and thus, they precipitate
out during dialysis. This process left behind the properly folded
molecules in solution that bound to UDP-agarose and were
enzymatically active. (FIG. 8) (Table IX). It is thought that the
SR-domain, like PEG-4000 and L-arginine, helped to solubilize the
folding intermediates, and hence enhanced the formation of both
native and misfolded-SRCD molecules. The presence of PEG-4000 and
L-arginine enhanced the solubilization of the folding intermediates
of SRCD-molecules even further. The misfolded SRCD proteins, in
contrast to the majority of CD-proteins, remained soluble even in
the absence of PEG-4000 and L-arginine. Therefore, the misfolded
SRCD-proteins were not removed as precipitates during dialysis.
Misfolded SRCD-proteins can be separated from properly folded
proteins through binding on UDP-agarose columns (Table IX). Thus
the SR-domain is thought to act as a solubilizing agent both for
the misfolded and folded catalytic domain. It is thought that the
increased solubility of SRCD-proteins is produced by preventing
aggregation of misfolded proteins. In this respect its mode of
action is thought to resemble the action of chaperone proteins. The
positive effect of the N-terminal stem region in the folding and
stability of the native protein is very useful for producing large
quantities of other galactosyltransferase family members.
[0065] The in vitro folding efficiency of bovine
.beta.(1,4)-galactosyltransferase I was further increased by
substituting the cysteine at amino acid position 342 with a
threonine (C342T). Analogous mutations can be made in
.beta.(1,4)-galactosyltransferase I enzymes from other
organisms.
[0066] It was determined that the wild-type bovine
SRCD.beta.4Gal-T1, folded and purified from inclusion bodies, was
cleaved at Ser96 within the stem region over a short period of
time. Therefore, to decrease degradation of bovine
SRCD.beta.4Gal-T1, the serine at amino acid position 96 was
exchanged with an Ala to produce S96A-SRCD.beta.4Gal-T1. After
folding and purification from bacterial inclusion bodies,
S96A-SRCD.beta.4Gal-T1 was found to be more stable over a long
period of time when compared to SRCD.beta.4Gal-T1, which did not
include the S96A mutation. S96A-SRCD.beta.4Gal-T1 was used within
some of the in vitro folding studies disclosed herein.
[0067] The in vitro folding efficiency of catalytic domains that
include a stem region in the presence of PEG-4000 and L-Arg was
about 50 percent and the solubility of the refolded product was
about 90 percent.
[0068] Accordingly, the invention includes stem regions from
members of the galactosyltransferase family that can be fused to a
catalytic domain of a galactosyltransferase to provide increased in
vitro folding of the catalytic domain. Such stem regions can be
readily determined based on amino acid sequence homology to the
bovine stem region and tested for the ability to promote folding of
a galactosyltransferase catalytic domain. The invention also
includes the mutants disclosed herein and their corresponding
analogs in other species.
[0069] General methods for isolating and folding inclusion bodies
containing galactosyltransferase catalytic domains have been
previously described (Ramakrishnan et al., J. Biol. Chem. 276:37665
(2001)). These methods may be used in conjunction with the stem
region of the invention, PEG4000, and L-Arg to increase the folding
efficiency of a galactosyltransferase catalytic domain. These
methods are described in the examples section herein.
IV. Nucleic Acid Segments Encoding Catalytic Domains of
.beta.(1.4)-galactosyltransferase I, Expression Cassettes that
Include the Nucleic Acid Segments, and Cells that Include the
Nucleic Acid Segments and Expression Cassettes.
[0070] The present invention provides isolated nucleic acid
segments that encode catalytic domains of
.beta.(1,4)-galactosyltransferase I having altered donor or
acceptor specificity. The present invention also provides nucleic
acid segments that encode amino acid segments that promote proper
folding of catalytic domains from galactosyltransferases, such as
.beta.(1,4)-galactosyltransferase I.
[0071] Nucleic acid sequences encoding human
.beta.(1,4)-galactosyltransferase I (SEQ ID NO: 8), as well as
other .beta.(1,4)-galactosyltransferases I from other organisms are
available. These nucleic acid sequences can be modified to encode
the catalytic domains and amino acid segments of the invention
through use of well-known techniques (Sambrook et al., Molecular
Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor
Press, Cold Spring Harbor, N.Y. (2001)). For example, a portion of
the nucleic acid sequence encoding human
.beta.(1,4)-galactosyltransferase I (SEQ ID NO: 8) can be inserted
into an expression vector such that an amino acid segment
corresponding to the catalytic domain of human
.beta.(1,4)-galactosyltransferase I (SEQ ID NO: 6) is expressed
upon transformation of a cell with the expression vector. In
another example, bovine .beta.(1,4)-galactosyltransferase I can be
altered to replace the tyrosine at amino acid position 289 with
leucine, isoleucine, or asparagine through use of site-directed
mutagenesis (Ramakrishnan et al., J. Biol. Chem., 277:20833
(2002)). Similar methods may be used to produce nucleic acid
segments encoding additional mutants and catalytic domains
described herein.
[0072] The nucleic acid segments of the invention may be optimized
for expression in select cells. Codon optimization tables are
available. Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, 1988.
[0073] The nucleic acid segments can be inserted into numerous
types of vectors. A vector may include, but is not limited to, any
plasmid, phagemid, F-factor, virus, cosmid, or phage in double or
single stranded linear or circular form which may or may not be
self transmissible or mobilizable. The vector can also transform a
prokaryotic or eukaryotic host either by integration into the
cellular genome or exist extrachromosomally (e.g. autonomous
replicating plasmid with an origin of replication).
[0074] Preferably the nucleic acid segment in the vector is under
the control of, and operably linked to, an appropriate promoter or
other regulatory elements for transcription in vitro or in a host
cell such as a eukaryotic cell or microbe, e.g. bacteria. The
vector may be a bi-functional expression vector which functions in
multiple hosts. In the case of genomic DNA, this may contain its
own promoter or other regulatory elements and in the case of cDNA
this may be under the control of a promoter or other regulatory
sequences for expression in a host cell.
[0075] Specifically included are shuttle vectors by which is meant
a DNA vehicle capable, naturally or by design, of replication in
two different host organisms, which may be selected from bacteria
and eukaryotic cells (e.g. mammalian, yeast or fungal).
[0076] The vector may also be a cloning vector which typically
contains one or a small number of restriction endonuclease
recognition sites at which nucleic acid segments can be inserted in
a determinable fashion. Such insertion can occur without loss of
essential biological function of the cloning vector. A cloning
vector may also contain a marker gene that is suitable for use in
the identification and selection of cells transformed with the
cloning vector. Examples of marker genes are tetracycline
resistance, hygromycin resistance or ampicillin resistance. Many
cloning vectors are commercially available (Stratagene, New England
Biolabs, Clonetech).
[0077] The nucleic acid segments of the invention may also be
inserted into an expression vector. Typically an expression vector
contains (1) prokaryotic DNA elements coding for a bacterial
replication origin and an antibiotic resistance gene to provide for
the amplification and selection of the expression vector in a
bacterial host; (2) regulatory elements that control initiation of
transcription such as a promoter; and (3) DNA elements that control
the processing of transcripts such as introns, transcription
termination/polyadenylation sequence.
[0078] Methods to introduce a nucleic acid segment into a vector
are well known in the art (Sambrook et al., 1989). Briefly, a
vector into which the nucleic acid segment is to be inserted is
treated with one or more restriction enzymes (restriction
endonuclease) to produce a linearized vector having a blunt end, a
"sticky" end with a 5' or a 3' overhang, or any combination of the
above. The vector may also be treated with a restriction enzyme and
subsequently treated with another modifying enzyme, such as a
polymerase, an exonuclease, a phosphatase or a kinase, to create a
linearized vector that has characteristics useful for ligation of a
nucleic acid segment into the vector. The nucleic acid segment that
is to be inserted into the vector is treated with one or more
restriction enzymes to create a linearized segment having a blunt
end, a "sticky" end with a 5' or a 3' overhang, or any combination
of the above. The nucleic acid segment may also be treated with a
restriction enzyme and subsequently treated with another DNA
modifying enzyme. Such DNA modifying enzymes include, but are not
limited to, polymerase, exonuclease, phosphatase or a kinase, to
create a polynucleic acid segment that has characteristics useful
for ligation of a nucleic acid segment into the vector.
[0079] The treated vector and nucleic acid segment are then ligated
together to form a construct containing a nucleic acid segment
according to methods known in the art (Sambrook, 2002). Briefly,
the treated nucleic acid fragment and the treated vector are
combined in the presence of a suitable buffer and ligase. The
mixture is then incubated under appropriate conditions to allow the
ligase to ligate the nucleic acid fragment into the vector. It is
preferred that the nucleic acid fragment and the vector each have
complimentary "sticky" ends to increase ligation efficiency, as
opposed to blunt-end ligation. It is more preferred that the vector
and nucleic acid fragment are each treated with two different
restriction enzymes to produce two different complimentary "sticky"
ends. This allows for directional ligation of the nucleic acid
fragment into the vector, increases ligation efficiency and avoids
ligation of the ends of the vector to reform the vector without the
inserted nucleic acid fragment.
[0080] Suitable procaryotic vectors include but are not limited to
pBR322, pMB9, pUC, lambda bacteriophage, m13 bacteriophage, and
Bluescript.RTM.. Suitable eukaryotic vectors include but are not
limited to PMSG, pAV009/A+, PMTO10/A+, pMAM neo-5, bacculovirus,
pDSVE, YIP5, YRP17, YEP. It will be clear to one of ordinary skill
in the art which vector or promoter system should be used depending
on which cell type is used for a host cell.
[0081] The invention also provides expression cassettes which
contain a control sequence capable of directing expression of a
particular nucleic acid segment of the invention either in vitro or
in a host cell. The expression cassette is an isolatable unit such
that the expression cassette may be in linear form and functional
in in vitro transcription and translation assays. The materials and
procedures to conduct these assays are commercially available from
Promega Corp. (Madison, Wis.). For example, an in vitro transcript
may be produced by placing a nucleic acid segment under the control
of a T7 promoter and then using T7 RNA polymerase to produce an in
vitro transcript. This transcript may then be translated in vitro
through use of a rabbit reticulocyte lysate. Alternatively, the
expression cassette can be incorporated into a vector allowing for
replication and amplification of the expression cassette within a
host cell or also in vitro transcription and translation of a
nucleic acid segment.
[0082] Such an expression cassette may contain one or a plurality
of restriction sites allowing for placement of the nucleic acid
segment under the regulation of a regulatory sequence. The
expression cassette can also contain a termination signal operably
linked to the nucleic acid segment as well as regulatory sequences
required for proper translation of the nucleic acid segment.
Expression of the nucleic acid segment in the expression cassette
may be under the control of a constitutive promoter or an inducible
promoter which initiates transcription only when the host cell is
exposed to some particular external stimulus.
[0083] The expression cassette may include in the 5'-3' direction
of transcription, a transcriptional and translational initiation
region, a nucleic acid segment and a transcriptional and
translational termination region functional in vivo and/or in
vitro. The termination region may be native with the
transcriptional initiation region, may be native with the nucleic
acid segment, or may be derived from another source. Numerous
termination regions are known in the art. Guerineau et al., Mol.
Gen. Genet., 262:141 (1991); Proudfoot, Cell, 64:671 (1991);
Sanfacon et al., Genes Dev., 5:141 (1991); Munroe et al., Gene,
91:151 (1990); Ballas et al., Nucleic Acids Res., 17:7891 (1989);
Joshi et al., Nucleic Acid Res., 15:9627 (1987).
[0084] The regulatory sequence can be a nucleic acid sequence
located upstream (5' non-coding sequences), within, or downstream
(3' non-coding sequences) of a coding sequence, and which
influences the transcription, RNA processing or stability, or
translation of the associated coding sequence. Regulatory sequences
can include, but are not limited to, enhancers, promoters,
repressor binding sites, translation leader sequences, introns, and
polyadenylation signal sequences. They may include natural and
synthetic sequences as well as sequences which may be a combination
of synthetic and natural sequences. While regulatory sequences are
not limited to promoters, some useful regulatory sequences include
constitutive promoters, inducible promoters, regulated promoters,
tissue-specific promoters, viral promoters and synthetic
promoters.
[0085] A promoter is a nucleotide sequence that controls expression
of the coding sequence by providing the recognition for RNA
polymerase and other factors required for proper transcription. A
promoter includes a minimal promoter, consisting only of all basal
elements needed for transcription initiation, such as a TATA-box
and/or initiator that is a short DNA sequence comprised of a
TATA-box and other sequences that serve to specify the site of
transcription initiation, to which regulatory elements are added
for control of expression. A promoter may be inducible. Several
inducible promoters have been reported (Current Opinion in
Biotechnology, 7:168 (1996)). Examples include the tetracycline
repressor system, Lac repressor system, copper-inducible systems,
salicylate-inducible systems (such as the PR1a system). Also
included are the benzene sulphonamide- (U.S. Pat. No. 5,364,780)
and alcohol- (WO 97/06269 and WO 97/06268) inducible systems and
glutathione S-transferase promoters. In the case of a multicellular
organism, the promoter can also be specific to a particular tissue
or organ or stage of development.
[0086] An enhancer is a DNA sequence which can stimulate promoter
activity and may be an innate element of the promoter or a
heterologous element inserted to enhance the level or tissue
specificity of a promoter. It is capable of operating in both
orientations (normal or flipped), and is capable of functioning
even when moved either upstream or downstream from the promoter.
Both enhancers and other upstream promoter elements bind
sequence-specific DNA-binding proteins that mediate their
effects.
[0087] The expression cassette can contain a 5' non-coding sequence
which is a nucleotide sequence located 5' (upstream) to the coding
sequence. It is present in the fully processed mRNA upstream of the
initiation codon and may affect processing of the primary
transcript to mRNA, stability of the mRNA, or translation
efficiency (Turner et al., Molecular Biotechnology, 3:225
(1995)).
[0088] The expression cassette may also contain a 3' non-coding
sequence which is a nucleotide sequence located 3' (downstream) to
a coding sequence and includes polyadenylation signal sequences and
other sequences encoding regulatory signals capable of affecting
mRNA processing or gene expression. The polyadenylation signal is
usually characterized by affecting the addition of polyadenylic
acid tracts to the 3' end of the mRNA precursor.
[0089] The invention also provides a construct containing a vector
and an expression cassette. The vector may be selected from, but
not limited to, any vector previously described. Into this vector
may be inserted an expression cassette through methods known in the
art and previously described (Sambrook et al., 1989). In one
embodiment, the regulatory sequences of the expression cassette may
be derived from a source other than the vector into which the
expression cassette is inserted. In another embodiment, a construct
containing a vector and an expression cassette is formed upon
insertion of a nucleic acid segment of the invention into a vector
that itself contains regulatory sequences. Thus, an expression
cassette is formed upon insertion of the nucleic acid segment into
the vector. Vectors containing regulatory sequences are available
commercially and methods for their use are known in the art
(Clonetech, Promega, Stratagene).
[0090] The expression cassette, or a vector construct containing
the expression cassette may be inserted into a cell. The expression
cassette or vector construct may be carried episomally or
integrated into the genome of the cell.
[0091] A variety of techniques are available and known to those
skilled in the art for introduction of constructs into a cellular
host. Transformation of bacteria and many eukaryotic cells may be
accomplished through use of polyethylene glycol, calcium chloride,
viral infection, phage infection, electroporation and other methods
known in the art. Other transformation methods are available to
those skilled in the art, such as direct uptake of foreign DNA
constructs (see EP 295959), techniques of electroporation (Fromm et
al. Nature (London), 319:791 (1986) or high velocity ballistic
bombardment with metal particles coated with the nucleic acid
constructs (Kline et al. Nature (London) 327:70 (1987), and U.S.
Pat. No. 4,945,050).
[0092] The selection of an appropriate expression vector will
depend upon the method of introducing the expression vector into
host cells. Typically an expression vector contains (1) prokaryotic
DNA elements coding for a bacterial origin of replication and an
antibiotic resistance gene to provide for the amplification and
selection of the expression vector in a bacterial host; (2) DNA
elements that control initiation of transcription, such as a
promoter; (3) DNA elements that control the processing of
transcripts, such as introns, transcription
termination/polyadenylation sequence; and (4) a reporter gene that
is operatively linked to the DNA elements to control transcription
initiation. Useful reporter genes include .beta.-galactosidase,
chloramphenicol acetyl transferase, luciferase, green fluorescent
protein (GFP) and the like.
V. Methods to Synthesize glucose-.beta.(1.4)-N-acetylglucosamine
Moieties, N-acetylgalactosamine-.beta.(1,4)-N-acetyl Glucosamine
Moieties: N-acetylgalactosamine-.beta.(1,4)-glucose moieties:
N-acetylglucosamine-.beta.(1.4)-N-acetylglucosamine Moieties:
mannose-.beta.(1,4)-N-acetylglucosamine Moieties; and
galactose-.beta.(1,4)-N-acetylglucosamine-6-SO.sub.3 Moieties.
[0093] Catalytic domains of the invention having altered donor and
acceptor specificity can be used to catalyze the linkage of
numerous sugars from a donor to numerous acceptor sugars. Linkage
of sugar derivatives can also achieved through use of the altered
catalytic domains of the invention due to their expanded donor and
acceptor specificity.
[0094] For example, the catalytic domains of section IA can be used
to catalyze the linkage of glucose and N-acetylglucosamine; the
catalytic domains of section IB can be used to catalyze the linkage
of N-acetylgalactosamine and N-acetylglucosamine; many of the
catalytic domains described herein can be used in association with
.alpha.-lactalbumin to catalyze linkage of a sugar to glucose, as
described in section IC; the catalytic domains of section ID can be
used to catalyze the linkage of N-acetylglucosamine and
N-acetylglucosamine, N-acetylgalactosamine to N-acetylglucosamine,
and mannose to N-acetylglucosamine; and the catalytic domains of
section IE can be used to catalyze the linkage of a donor and an
acceptor having a bulky side-group, such as linking galactose to
N-acetylglucosamine-6-SO.sub.3.
[0095] Acceptors may be free in solution or linked to another
molecule. For example, an acceptor may be linked to a protein,
another sugar, a sugar derivative, and the like. An acceptor may
also be linked to a solid support that provides a platform to which
donors may be added sequentially to form oligosaccharides, and
derivatives thereof, having a specified sequence.
[0096] Generally, the linkage between a donor and an acceptor is
accomplished by incubating a catalytic domain of the invention with
a desired donor and a desired acceptor under conditions of
appropriate temperature, pH, and divalent metal concentration to
allow linkage of the donor to the acceptor.
[0097] For example, the galactose and N-acetylgalactosyltransferase
activity of .beta.(1,4)-galactosyltransferase I can be determined
using the following assay conditions. A 100 .mu.l incubation
mixture containing 50 mM .beta.-benzyl-GlcNAc, 10 mM MnCl.sub.2, 10
mM Tris-HCl (pH 8.0), 500 .mu.M UDP-Gal or UDP-GalNAc, and 20 ng of
.beta.(1,4)-galactosyltransferase I can be incubated at 37.degree.
C. for 10 minutes to promote coupling of a donor sugar to an
acceptor sugar. While these conditions are provided as an example,
it is understood that many other conditions may be used to
chemically link a donor to an acceptor using the altered catalytic
domains of the invention.
VI. Methods to Prepare Oligosaccharides.
[0098] The invention provides methods to synthesize
oligosaccharides, especially oligosaccharides having preselected
sequences though use of the altered catalytic domains of the
invention. Generally, the methods involve the sequential addition
of a sugar, or derivative thereof, to the end of a growing
oligosaccharide chain. Such methods have been described using
enzymes other than those of the invention (U.S. Pat. No.
6,284,493).
[0099] Briefly, a donor and an acceptor may be incubated with an
altered catalytic domain of the invention under conditions that
allow the donor to be linked to the acceptor. These conditions are
described in the examples section herein.
[0100] In one example, the donor and the acceptor may be combined
with a catalytic domain of the invention in solution. The solution
is then incubated to allow the donor to be linked to the acceptor.
The newly linked molecule may be isolated and then added to a
second solution containing a second donor and a second transferase
enzyme. This cycle may be repeated with a specific donor added at
each cycle such that an oligosaccharide having a specific sequence
is produced.
[0101] In another example, an acceptor may be linked to a solid
support. The solid support may then be immobilized in a structure
such as a column or a tray. A donor and a catalytic domain of the
invention can then be incubated with the immobilized acceptor under
conditions that allow the donor to be linked to the acceptor. The
solid support is then washed to remove any unlinked donor and
catalytic domain present. A second donor and a second transferase
enzyme can then be added and incubated under conditions that allow
the donor to be linked to the acceptor. This cycle can be repeated
to allow for the rapid and large-scale production of
oligosaccharides having defined sequences. In addition, this method
may be readily adapted for use in an automated system. This system
may be used without the need for protecting groups on the acceptor
or the donor due to the use of enzymes that catalyze the linkage of
a given donor to a given acceptor. Accordingly, an advantage of
this method is that mild reaction conditions may be used that do
not damage the growing oligosaccharide chain. Another advantage of
the method is the short cycling time required to add monomers onto
the growing oligosaccharide chain due to the lack of a need to
protect and deprotect the growing oligosaccharide chain.
[0102] Other methods for using the catalytic domains of the
invention to synthesize sequences of predetermined oligosaccharides
and derivatives thereof may also be used. However, these methods
will utilize an galactosyltransferase having an altered acceptor
site, an altered donor site, or altered donor and acceptor
sites.
VII. Methods to Increase the Immunogenicity of an Antigen.
[0103] The invention provides methods to increase the
immunogenicity of an antigen. Generally, the methods involve
incubating an antigen with a catalytic domain of the invention such
that a sugar is transferred from a donor to an acceptor through the
action of a catalytic domain.
[0104] The methods of the invention may be used in association with
nearly any acceptor containing material against which an immune
response is desired. For example, sugars may be transferred from a
donor to an acceptor that is linked to a whole cell. The cell can
then be killed through irradiation or chemical means and
administered to an animal to elicit an immune response. Cell
membranes may be used in a similar manner. Methods to create an
immune response against cells and cell membranes are described in
U.S. Pat. No. 6,361,775.
[0105] The immunogenicity of a virus or subunit thereof may be
increased according to the methods of the invention and used as an
improved vaccine. For example, for a virus that contains a
glycoprotein as a component of the virion, one or more sugars may
be added to the glycoprotein by propagation of the virus in a cell
that expresses a catalytic domain of the invention. Alternatively,
one or more sugars may be directly added to the glycoprotein using
a catalytic domain of the invention. Furthermore, there exist
viruses without envelopes that contain complex carbohydrates.
Sugars may be added onto these carbohydrates through use of a
catalytic domain of the invention.
[0106] Viral subunits may be obtained from virions using
biochemical methods or they can be expressed by recombinant means
in suitable eukaryotic cells. Methods of expressing viral subunits
are common in the art. These methods may vary according to the type
of virus used. For example, methods of expressing viral subunits
are described in the following articles and in the references cited
therein: Possee, Virus Research. 5:43 (1986); Kuroda et al., EMBO
J., 5: 1359 (1986); Doerfler, Curr. Topics Microbiol. Immunol.,
131:51 (1986); Rigby, J. Gen. Virol. 64:255 (1983); Mackett et al.,
In: DNA Cloning, A Practical Approach, Vol II, Ed. D. M. Glover,
IRL Press, Washington, D.C. (1985); Rothestein, In: DNA Cloning, A
Practical Approach, Supra (1985); Kinney et al., J. Gen. Virol.
69:3005 (1988); Panical et al., Proc. Natl. Acad. Sci., 80:5364
(1983); Small et al., In: Vaccinia Viruses as Vectors for Vaccine
Antigens, pp. 175-178, Ed. J. Quinnan, Elsevier, N.Y (1985).
[0107] Viruses for which vaccines are currently available and whose
immunogenicity can be improved according to the methods of the
invention include influenza virus (Orthomyxovirus); rabies virus
(Rhabdovirus); hepatitis B virus (Hepadnavirus); eastern, western
and Venezuelan equine encephalitis virus (Togavirus/Alphavirus
genus); and, Japanese encephalitis virus, tick-borne encephalitis
virus and Russian spring-summer encephalitis virus (Flavivirus);
Rift Valley fever virus (Bunyavirus) (reviewed: Melnick, In: High
Technology Route to Virus Vaccines Ed. Dreesman, Bronson and
Kennedy, American Society for Microbiology, Washington, D.C.
(1985); Ogra et al., Prog. Med. Virol., 37:156 (1990)). Viruses for
which vaccines are not yet commercially available but which may
also be useful when treated according to the methods of the
invention include, but are not limited to human immunodeficiency
and human T-cell leukemia viruses (Retrovirus); respiratory
syncytial virus and other paramyxoviruses (Paramyxovirus); herpes
simplex viruses types 1 and 2, varicella zoster virus,
cytomegalovirus and other herpes viruses (Herpesviruses); dengue
virus and Saint Louis encephalitis virus (Flavivirus); hantaan
virus (Bunyavirus); Lassa virus (Arenavirus); and, rotavirus
(Reovirus). In addition, there exist viral vaccines comprising live
attenuated viruses the administration of which to humans is
associated with some measure of risk of mild to severe side
effects. It is now possible according to the methods of the
invention to enhance the immunogenicity of killed virus vaccines
which may serve to reduce the use of their live, more
risk-associated counterparts. Thus, vaccines which currently
comprise live virus, such as measles virus, mumps virus,
rubellavirus etc. are all encompassed by the invention.
[0108] Vaccines are prepared by suspension of a suitable
concentration of an antigen that has been linked to a sugar,
through the action of a catalytic domain of the invention, in a
pharmaceutically acceptable carrier. The composition of the carrier
will depend upon the type of vaccine and the route of
administration and will be readily apparent to one skilled in the
art. The vaccine may be administered in a dose of 10.sup.2 to
10.sup.9 cells per dose (in the case of whole cells) or a similar
equivalent of cell membranes. In the case of viral vaccines, virus
may be administered in dose ranging from 1 .mu.g to 50 mg of virus
per dose, or a similar equivalent of subunit. Determination of the
appropriate dosage of vaccine will be apparent to one of skill in
the art and will depend upon the antigens comprising the vaccine,
the age of the patient and their general and immunological
health.
[0109] In order to improve the efficacy of vaccines prepared
according to the methods of the invention, patients may be
pretreated with adjuvant at the site of vaccination several days
prior to administration of vaccine. Pretreatment with adjuvant
serves to induce migration of macrophages to the site of
inoculation, thereby enhancing the rate of phagocytosis of treated
antigens. In the case of viral vaccines, patients may be treated
with adjuvant and vaccine simultaneously. Adjuvants suitable for
this purpose include aluminum hydroxide and like adjuvants.
[0110] Vaccines are administered to a mammal, particularly a human,
either subcutaneously, intramuscularly, orally, intravenously,
intradermally, intranasally or intravaginally. Prior to oral
administration, the vaccine can be mixed with a solution containing
a sufficient amount of sodium bicarbonate or other suitable
compound capable of neutralizing stomach acid (approximately 2
grams). Alternatively, the vaccine usually in lyophilized form, can
be formulated as tablets which are treated with a coating capable
of resisting stomach acid.
TABLE-US-00001 TABLE I Exemplary Amino Acid Sequences SEQ ID
Accession NO Number Description Amino Acid Sequence 3 BAA06188
Human MRLREPLLSRSAAMPGASLQRACRLLVAVCA .beta.(1,4)galactosyl-
LHLGVTLVYYLAGRDLSRLPQLVGVSTPLQG transferase
GSNSAAAIGQSSGDLRTGGARPPPPLGASSQ (398 AA)
PRPGGDSSPVVDSGPGPASNLTSVPVPHTTA LSLPACPEESPLLVGPMLIEFNMPVDLELVA
KQNPNVKMGGRYAPRDCVSPHKVAIIIPFRN RQEHLKYWLYYLHPVLQRQQLDYGIYVINQA
GDTIFNRAKLLNVGFQEALKDYDYTCFVFSD VDLIPMNDHNAYRCFSQPRHISVAMDKFGFS
LPYVQYFGGVSASSKQQFLTINGFPNNYWGW GGEDDDIFNRLVFRGMSISRPNAVVGTCRMI
RHSRDKKNEPNPQRFDRIAHTKETMLSDGLN SLTYQVLDVQRYPLYTQITVDIGTPS 4 A33396
Mouse MRFREQFLGGSAAMPGATLQRACRLLVAVC .beta.(1,4)galactosyl-
ALHLGVTLVYYLSGRDLSRLPQLVGVSSTLQ transferase
GGTNGAAASKQPPGEQRPRGARPPPPLGVSP (399 AA)
KPRPGLDSSPGAASGPGLKSNLSSLPVPTTT GLLSLPACPEESPLLVGPMLIDFNIAVDLEL
LAKKNPEIKTGGRYSPKDCVSPHKVAIIIPF RNRQEHLKYWLYYLHPILQRQQLDYGIYVIN
QAGDTMFNRAKLLNIGFQEALKDYDYNCFVF SDVDLIPMDDRNAYRCFSQPRHISVAMDKFG
FSLPYVQYFGGVSALSKQQFLAINGFPNNYW GWGGEDDDIFNRLVHKGMSISRPNAVVGRCR
MIRHSRDKKNEPNPQRFDRIAHTKETMRFDG LNSLTYKVLDVQRYPLYTQITVDIGTPR 5
S05018 Bovine MKFREPLLGGSAAMPGASLQRACRLLVAVC .beta.(1,4)galactosyl-
ALHLGVTLVYYLAGRDLRRLPQLVGVHPPLQ transferase
GSSHGAAAIGQPSGELRLRGVAPPPPLQNSS (402 AA)
KPRSRAPSNLDAYSHPGPGPGPGSNLTSAPV PSTTTRSLTACPEESPLLVGPMLIEFNIPVD
LKLIEQQNPKVKLGGRYTPMDCISPHKVAII ILFRNRQEHLKYWLYYLHPMVQRQQLDYGIY
VINQAGESMFNRAKLLNVGFKEALKDYDYNC FVFSDVDLIPMNDHNTYRCFSQPRHISVAMD
KFGFSLPYVQYFGGVSALSKQQFLSINGFPN NYWGWGGEDDDIYNRLAFRGMSVSRPNAVIG
KCRMIRHSRDKKNEPNPQRFDRIAHTKETML SDGLNSLTYMVLEVQRYPLYTKITVDIGTPS 6
Human RDLSRLPQLVGVSTPLQGGSNSAAAIGQSSG Stem Region of
DLRTGGARPPPPLGASSQPRPGGDSSPVVDS .beta.(1,4)galactosyl-
GPGPASNLTSVPVPHTTALSLPACPEESPLL transferase VGPMLIEFNMPVDLELVAKQ 7
Bovine RDLRRLPQLVGVHPPLQGSSHGAAAIGQPSG Stem Region of
ELRLRGVAPPPPLQNSSKPRSRAPSNLDAYS .beta.(1,4)galactosyl-
HPGPGPGPGSNLTSAPVPSTTTR transferase 8 Human Catalytic
SLPACPEESPLLVGPMLIEFNMPVDLELVAK Domain of
QNPNVKMGGRYAPRDCVSPHKVAIIIPFRNR .beta.(1,4)galactosyl-
QEHLKYWLYYLHPVLQRQQLDYGIYVINQAG transferase
DTIFNRAKLLNVGFQEALKDYDYTCFVFSDV DLIPMNDHNAYRCFSQPRHISVAMDKFGFSL
PYVQYFGGVSASSKQQFLTINGFPNNYWGWG GEDDDIFNRLVFRGMSISRPNAVVGTCRMIR
HSRDKKNEPNPQRFDRIAHTKETMLSDGLNS LTYQVLDVQRYPLYTQITVDIGTPS 9 Bovine
Catalytic SLTACPEESPLLVGPMLIEFNIPVDLKLIEQ Domain of
QNPKVKLGGRYTPMDCISPHKVAIIILFRNR .beta.(1,4)galactosyl-
QEHLKYWLYYLHPMVQRQQLDYGIYVINQAG transferase
ESMFNRAKLLNVGFKEALKDYDYNCFVFSDV DLIPMNDHNTYRCFSQPRHISVAMDKFGFSL
PYVQYFGGVSALSKQQFLSINGFPNNYWGWG GEDDDIYNRLAFRGMSVSRPNAVIGKCRMIR
HSRDKKNEPNPQRFDRIAHTKETMLSDGLNS LTYMVLEVQRYPLYTKITVDIGTPS
TABLE-US-00002 TABLE II Exemplary Nucleic Acid Sequence SEQ ID
Accession NO Number Description Nucleic Acid Sequence 10 D29805
Human ATGAGGCTTCGGGAGCCGCTCCTGAGCCGGA .beta.3(1,4)galactosyl-
GCGCCGCGATGCCAGGCGCGTCCCTACAGCG transferase
GGCCTGCCGCCTGCTCGTCGCCGTCTGCGCT CTGCACCTTGGCGTCACCCTCGTTTACTACC
GCTGGCCGCGACCTGAGCCGCCTGCCCCAAC TGGTCGGAGTCTCCACACCGCTGCAGGGCGG
GTCGAACAGTGCCGCCGCCATCGGGCAGTCC TCCGGGGACCTCCGGACCGGAGGGGCCCGGC
CGCCGCCTCCTCTAGGCGCCTCCTCCCAGCC GCGCCCGGGTGGCGACTCCAGCCCAGTCGTG
GATTCTGGCCCTGGCCCCGCTAGCAACTTGA CCTCGGTCCCAGTGCCCCACACCACCGCACT
GTCGCTGCCCGCCTGCCCTGAGGAGTCCCCG CTGCTTGTGGGCCCCATGCTGATTGAGTTTA
ACATGCCTGTGGACCTGGAGCTCGTGGCAAA GCAGAACCCAAATGTGAAGATGGGCGGCCGC
TATGCCCCCAGGGACTGCGTCTCTCCTCACA AGGTGGCCATCATCATTCCATTCCGCAACCG
GCAGGAGCACCTCAAGTACTGGCTATATTAT TTGCACCCAGTCCTGCAGCGCCAGCAGCTGG
ACTATGGCATCTATGTTATCAACCAGGCGGG AGACACTATATTCAATCGTGCTAAGCTCCTC
AATGTTGGCTTTCAAGAAGCCTTGAAGGACT ATGACTACACCTGCTTTGTGTTTAGTGACGT
GGACCTCATTCCAATGAATGATCATAATGCG TACAGGTGTTTTTCACAGCCACGGCACATTT
CCGTTGCAATGGATAAGTTTGGATTCAGCCT ACCTTATGTTCAGTATTTTGGAGGTGTCTCT
GCTTCAAGTAAACAACAGTTTCTAACCATCA ATGGATTTCCTAATAATTATTGGGGCTGGGG
AGGAGAAGATGATGACATTTTTAACAGATTA GTTTTTAGAGGCATGTCTATATCTCGCCCAA
ATGCTGTGGTCGGGACGTGTCGCATGATCCG CCACTCAAGAGACAAGAAAAATGAACCCAAT
CCTCAGAGGTTTGACCGAATTGCACACACAA AGGAGACAATGCTCTCTGATGGTTTGAACTC
ACTCACCTACCAGGTGCTGGATGTACAGAGA TACCCATTGTATACCCAAATCACAGTGGACA
TCGGGACACCGAGCTAG
Example 1
General Expression, Mutagenesis, Folding, and Purification of
Catalytic Domains of Bovine .beta.(1,4)-galactosyltransferase I
Materials and Methods
[0111] Bacterial growth and plasmid transformations were performed
using standard procedures (Ausubel et al., Current Protocols in
Molecular Biology, Greene Publishing Associates and
Wiley-Interscience, New York (1987)). The plasmid pEGT-d129, which
encodes the catalytic domain (residues 130-402) of bovine
.beta.(1,4)-galactosyltransferase I, was used for mutation.
Site-directed mutagenesis was performed using a CLONTECH
site-directed mutagenesis transformer kit. The transformation
mixture contained the template pEGT-d129, a selection primer, and a
mutagenic primer for creation of a desired mutant. Mutants were
screened for the incorporated mutations by looking for changes in
restriction enzyme digestion patterns and confirmed by DNA
sequencing. The positive clones were transformed into
B834(DE3)pLysS cells. The primers were synthesized by the Molecular
Technology Laboratory, NCI-Frederick.
[0112] The expression and purification of the inclusion bodies were
carried out as described previously (Ausubel et al., Current
Protocols in Molecular Biology, Greene Publishing Associates and
Wiley-Interscience, New York (1987)). The inclusion bodies were
S-sulfonated by dissolving in 5 M GdnHCl, 0.3 M sodium sulfite, and
the addition of di-sodium 2-nitro-5-thiosulfobenzoate to a final
concentration of 5 mM. The sulfonated protein was precipitated by
dilution with water, and the precipitate was washed thoroughly.
[0113] The sulfonated protein was re-dissolved in 5 M GdnHCl to a
protein concentration of 1 mg/ml (1.9-2.0 optical density at 275
nm). The protein solution was diluted 10-fold, in 10 portions, in a
folding solution to give a final concentration of 100 .mu.g/ml
.beta.(1,4)-galactosyltransferase I, 0.5 M GdnHCl, 50 mM Tris-HCl,
5 mM EDTA, 4 mM cysteamine, and 2 mM cystamine, pH 8.0 at 4.degree.
C. It was left at 4.degree. C. for 48 h to allow the protein to
fold and then dialyzed against 3.times.4 liters of water containing
50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 4 mM cysteamine, and 2 mM
cystamine at 4.degree. C. to remove GdnHCl. Any protein that
precipitated during dialysis was removed by centrifugation, and the
supernatant was concentrated. Typically, when 100 mg of sulfonated
protein was folded in a 1-liter folding solution, it yields 2-5 and
10-12 mg of the active, soluble, and pure wild-type
.beta.(1,4)-galactosyltransferase I catalytic domain, or mutant
thereof. The folded proteins were purified further on an LA-agarose
column (Sigma). The final, purified protein had a specific activity
that was slightly higher than that of purified milk
.beta.(1,4)-galactosyltransferase I.
[0114] Site-directed Mutagenesis of amino acid position 289 of the
bovine .beta.(1,4)-galactosyltransferase I: Site-directed
mutagenesis was performed using the PCR method. Construction of the
mutants was done using plasmid pEGT-d129 as the template; this
contains a BamH1/EcoRI fragment inserted into pEGT23a vector,
coding for the residues 130-402 of bovine
.beta.(1,4)-galactosyltransferase I, and has a Cys-342 to Thr
mutation.
[0115] The mutation primers corresponding to the upper DNA strand
are: Y289L, 5'-CCTTACGTGCAATTGTTTGGAGGTGTCTCTGCTCTAAGTAAA-3' (SEQ
ID NO: 11) and 5'-GACACCTCCAAACAATTGCACGTAAGGTAGGCTAAA-3' (SEQ ID
NO: 12); Y289I, 5'-CTACCTTACGTGCAGATCTTTGGAGGTGTCTCTGCTCTAAG-3'
(SEQ ID NO: 13) and 5'-GACACCTCCAAAGATCTGCACGTAAGGTAGGCTAATCCAA-3'
(SEQ ID NO: 14); Y289N,
5'-GGATTAGCCTACCATATGTGCAGAATTFTGGAGGTGTCTCT-3' (SEQ ID NO: 15) and
5'-AGAGACACCTCCAAAATTCTGCACATCTGGTAGGCTAAATCC-3' (SEQ ID NO: 16).
Typically, the entire Gal-T1 DNA was PCR-amplified as two fragments
using the terminal cloning primers and two mutagenesis primers. The
fragments were then cut with the restriction enzymes MfeI, BglII,
and NdeI for Y289L, Y286I, and Y286N mutants, respectively, and
ligated. The full Gal-T1 DNA with the mutation was amplified from
the ligation mixture using the cloning primers and then inserted
into the pET28a vector. Mutants were screened for the incorporated
mutations, based on alterations in the restriction enzyme digestion
patterns, and then sequenced. The positive clones were transformed
into B834(DES)pLys8 cells as described previously (Ramakrishnan et
al., J. Biol. Chem., 270, 87665-376717 (2001)). The mutant proteins
were expressed and purified according to the published method
(Ramakrishnan et al., J. Biol. Chem., 270, 87665-376717
(2001)).
[0116] Gal-T and GalNAc-T Enzyme Assays: The protein concentrations
were measured using the Bio-Rad protein assay kit, based on the
method of Bradford and further verified on SDS gel. An in vitro
assay procedure for the Gal-T1 has been reported previously
(Ramakrishnan et al., J. Biol. Chem., 270, 87665-376717 (2001)).
The activities were measured using UDP-Gal or UDP-GalNAc as sugar
nucleotide donors, and GlcNAc and Glc as the acceptor sugars. For
the specific activity measurements, a 100-.mu.l incubation mixture
containing 50 mM .beta.-benzyl-GlcNAc, 10 mM MnCl.sub.2, 10 mM
Tris-HCl, pH 8.0, 500 .mu.M UDP-Gal or UDP-GalNAc, 20 ng of Gal-T1,
and 0.5 .mu.Cl of [.sup.3H]UDP-Gal or [.sup.3H]UDP-GalNAc was used
for each Gal-T or GalNAc-T reaction. The incubation was carried out
at 37.degree. C. for 10 min. The reaction was terminated by adding
200 .mu.l of cold 50 mM EDTA, and the mixture was passed through a
0.5-ml bed volume column of AG1-X8 cation resin (Bio-Rad) to remove
any unreacted [.sup.3H]UDP-Gal or [.sup.3H]UDP-GalNAc. The column
was washed successfully with 300, 400, and 500 .mu.l of water, and
the column flow-through was diluted with Biosafe scintillation
fluid; radioactivity was measured with a Beckman counter. A
reaction without the acceptor sugar was used as a control. A
similar assay was carried out to measure the GalNAc-T activity with
Glc and other acceptors in the presence of 50 .mu.M bovine LA
(Sigma).
[0117] Studies for Determining the Kinetic Constants: The true
K.sub.m of the donor (K.sub.A) and of the acceptor (K.sub.B), the
dissociation constant of the donor, K.sub.i(a), and k.sub.cat were
obtained using two-substrate analyses and the primary plots of five
concentrations of donor (UDP-Gal or UDP-GalNAc) and five
concentrations of acceptor, and the corresponding secondary plots
of the intercepts and slopes. Initial rate conditions were linear
with respect to time. A suitable range of donor and acceptor
concentrations were chosen, which allowed an accurate
Michaelis-Menten plot to be derived. The data were also analyzed
for a general two-substrate system using the following equations
(Zhang et al., Glycobiology, 9:815-822 (1999)) with the software
EnzFitter, a Biosoft nonlinear curve-fitting program for
Windows.
v = V max [ A ] [ B ] K i ( a ) K B + K A [ A ] + K B [ B ] + [ A ]
[ B ] Equation 1 v = V max [ A ] [ B ] K A [ A ] + K B [ B ] + [ A
] [ B ] Equation 2 ##EQU00001##
Here v is the initial velocity and the rate equation for sequential
symmetrical initial velocity pattern associated with Equation 1, an
ordered or random equilibrium mechanism in which substrate A
dissociates well from the E-S complex with a dissociation constant
of K.sub.i(a). Equation 2 is for asymmetric initial velocity
pattern for a double displacement or "ping-pong" mechanism. The
kinetic parameters K.sub.A, K.sub.B, K.sub.i(a), and V.sub.max,
were obtained from the fitted curves using the above rate
equations. The graphical method and EnzFitter program gave very
similar kinetic parameters. In the N-acetylgalactosamine
transferase (GalNAc-T) assay, the maximum substrate concentrations
used for UDP-GalNAc and GlcNAc were 1 and 200 mm, respectively.
However, in the galactose transferase (Gal-T) assay, because of the
limited solubility of GlcNAc in water, the concentration of GlcNAc
was limited to no more than 400 mM (which is 2-fold higher than its
K.sub.m value), whereas up to 300 .mu.M UDP-Gal were used.
[0118] .sup.1H NMR Spectroscopy of the Products of GalNAc-T
Activity: The reaction was carried out in a total volume of 1 ml
that contained 100 .mu.g of Y289L mutant plus 10 mM each
triethanolamine-HCl, pH 8.0, UDP-GalNAc, GlcNAc, and MnCl.sub.2 at
37.degree. C. for 48 h. The mixture was first passed through a 1-ml
Chelex 100 column and then through a 1-ml cationic column
containing AG1-X8 resin (200-400-mesh). The disaccharide product in
the flow-through from 4 bed volumes was pooled and freeze-dried.
The product was finally dissolved in 400 .mu.l of D.sub.2O, and its
.sup.1H NMR spectrum was obtained in a 400-MHz NMR spectrometer.
All the NMR spectra were recorded in the Analytical Chemistry
Laboratory, NCI-Frederick (Frederick, Md.).
Results
[0119] Comparison of Gal-T and GalNAc-T Catalytic Activities of
Wild-type and Gal-T1 Mutants: The mutants Y289L, Y289N, and Y289I
exhibited both galactose transferase and
N-acetylgalactosyltransferase activities (Table III and FIG. 1).
The mutants Y289L and Y289N exhibited equally strong galactose
transferase and N-acetylgalactosyltransferase activities. The
specific N-acetylgalactosyltransferase activity of the mutant Y289I
at the substrate concentration used was half that of Y289L (Table
III). The Y289I mutant exhibited a slightly weaker affinity to
UDP-GalNAc (FIG. 1). The Asn substitution exhibited GalNAc-T
activity as well as the Leu substitution. Wild-type-Gal-T1 exhibits
a very low glucosyltranferase activity, but no
N-acetylglucosaminyltransferase activity (Berliner and Robinson,
Biochemistry, 21:6840-68433 (1982); Palmin and Hindaganl,
Glycobiology, 1:205-209 (1991); Ramakrishnan et al., J. Biol.
Chem., 270:87665-37671 (2001)). On the contrary, the mutants
exhibited reasonable N-acetylglucosaminyltransferase activity
(Table III) where they transfer N-acetylglucosamine from
UDP-N-acetylglucosamine to the acceptor N-acetylglucosamine but do
not exhibit glucosyltranferase activity. In this
N-acetylglucosaminyltransferase activity the initial product, the
disaccharide GlcNAc .beta.1,4-GlcNAc, itself is an acceptor for the
enzyme, thus producing tri- and longer chain saccharides.
TABLE-US-00003 TABLE III Specific activities of the catalytic
domain of Gal-T1 (residues 130-402) with the C342T mutation and the
Tyr-289 mutants UDP-galactose .fwdarw. GlcNAc UDP-GalNAc .fwdarw.
GlcNAc UDP-GlcNAc .fwdarw. GlcNAc Gal-T activity GalNAc-T activity
GlcNAc-T activity Enzyme pmol/min/ng pmol/min/ng pmol/min/ng C342T
11.8 0.1 0 Y289L_C342T 16.2 27.3 8.2 Y289I_C342T 8.8 14.7 6.2
Reactions were performed under saturating conditions of all
substrates (assay conditions are described herein). In these
reactions, the concentration for the donors was 500 .mu.M and the
acceptor .beta.-benzyl-GlcNAc was 25 mM. In the Gal-T reaction of
C342T, the acceptor concentration was 10 mM, because at 25 mM it
showed inhibition. Because Y289N rapidly undergoes denaturization,
reliable data could not be obtained.
[0120] Because the Y289L variant of Gal-T1 exhibits both Gal-T and
GalNAc-T enzyme activities with equal efficiencies, double
substrate kinetic studies were carried out to determine the kinetic
constants for both donor and acceptor molecules. The kinetic data
from both reactions fit best to Equation 2, with a zero K.sub.i(a)
value, describing an asymmetric initial velocity pattern for a
double-displacement or "ping-pong" mechanism. The Y289L mutant in
the Gal-T reaction showed nearly a 20-fold higher K.sub.m for the
acceptor than the wild-type Gal-T1 (Table IV). The catalytic
constant (k.sub.cat) in the Gal-T1 reaction is comparable with the
wild type, but it is nearly 3-5-fold higher in the GalNAc reaction
(Table IV).
TABLE-US-00004 TABLE IV Kinetic parameters for the donor and the
acceptor substrates by Y289L_C342T mutant in the Gal-T and the
GalNAc-T catalytic reactions GalNAc-T UDP-N- Gal-T UDP-galactose
.fwdarw. GlcNAc acetylgalactosamine .fwdarw. GlcNAc K.sub.A K.sub.B
K.sub.cat. K.sub.A K.sub.B K.sub.cat. Mutant .mu.M MM -- .mu.M mM
-- C342T 98 (6) 11(1) 14 ND ND ND Y289L_C342T 75 (1) 198(1) 8.5 854
(8) 51 (1) 4.0 ND, not determined
[0121] Because the Y289L mutant exhibits equally high GalNAc-T
activity as it does Gal-T activity, the disaccharide product from
this reaction was purified and analyzed by .sup.1H NMR spectroscopy
(FIG. 4). The NMR results demonstrated that the mutant Y289L
transfers GalNAc from UDP-GalNAc to GlcNAc, forming .beta.1-4
linkage between GalNAc and GlcNAc.
[0122] A Point Mutation in the Codon for Amino Acid 289 Can Convert
the Enzyme with Dual Property: In humans, the .beta.4Gal-T family
has seven members (Gal-T1 to -T7), each with high sequence identity
within their catalytic domain (Lo et al., Glycobiology, 8:517-526
(1998); Nomura et al., J. Biol. Chem., 273:13570-13577 (1998)).
These family members transfer Gal from UDP-Gal to different sugar
acceptors. For example, Gal-T6 transfers Gal to Glc of
Glc-ceramide, whereas Gal-T7 transfers Gal to xylose (Nomura et
al., J. Biol. Chem. 273:13570-13577 (1998)). Among these seven
members, four members, Gal-T1 to Gal-T4, have a Tyr residue at
position 287 (which corresponds to Tyr-289 in bovine Gal-T1),
whereas Gal-T5 and Gal-T6 have a Phe residue.
TABLE-US-00005 TABLE V Codon usage for the amino acid at position
289 among the Gal-T1 family members Enzyme Codon Amino acid at
position 289 Bovine Gal-T1 TAT Tyr Human Gal-T1 TAT Tyr Human
Gal-T2 TAC Tyr Human Gal-T3 TAC Tyr Human Gal-T4 TAT Tyr Human
Gal-T5 TTT Phe Human Gal-T6 TTC Phe Human Gal-T7 TAC Tyr In human
Gal-T1, the corresponding amino acid is at position 287. The second
and third nucleotides of the codon show variations among the family
members, while strictly conserving the first nucleotide. Mutation
of the first nucleotide of the codon (shown in bold) to either A or
C would result in a Leu or Asn residue instead of Tyr, and such a
mutant would exhibit dual enzymatic activities.
[0123] Role of .alpha.-Lactalbumin (LA) in the GalNAc-T Activity of
the wt-Gal-T1 and the Y289L Mutant: LA enhances the transfer of
GalNAc from UDP-GalNAc to GlcNAc by Gal-T1 with .about.0.1% of its
Gal-T efficiency, a situation similar to glucosyltransferase
activity (Glc-T) of Gal-T1, where LA increases this activity from
0.3 to 10% (Ramakrishnan et al., J. Biol. Chem., 270:87665-37671
(2001)). LA plays a kinetics role in stimulating Gal-T1 to transfer
Glc from the UDP-Glc to GlcNAc (Ramakrishnan et al., J. Biol.
Chem., 270:87665-37671 (2001); Ramakrishnan et al., Biochem
Biophys. Res. Commun., 291:1113-1118 (2002)). The activity is
enhanced by approximately 1%. This level of activity was determined
using the donor concentration of the unlabeled UDP-sugar, and only
a small amount of .sup.3H-labeled UDP-sugar. Use of this protocol
allowed for accurate calculation based on the amount of unlabeled
UDP-sugar used.
[0124] Unlike wt-Gal-T1, where LA stimulates the transfer of GalNAc
only to GlcNAc and not to Glc (Do et al., Biol. Chem.,
276:18447-18451 (1995)), the Tyr-289 mutants (Y289L, Y289N, and
Y289I) in the presence of LA transfer GalNAc preferably to Glc
rather than to GlcNAc. This property is quite similar to LS
activity in which the wt-Gal-T1 in the presence of LA transfers Gal
to Glc instead to GlcNAc (Table VI). Furthermore, like wt-Gal-T1,
these mutants also transfer Gal to Glc in the presence of LA. For
example, the N-acetylgalactosaminyltransferase activity in the
bovine mammary gland extracts with a similar catalytic property has
been identified (Van den Nieuwenhof et al., FEBS Lett., 459:377-380
(1999)). This enzyme also transfers GalNAc from UDP-GalNAc to
GlcNAc in the absence of LA, whereas in the presence of LA it
transfers GalNAc to Glc instead to GlcNAc.
TABLE-US-00006 TABLE VI GalNAc-T catalytic activity of Y289L mutant
in the absence and the presence of LA on various acceptor
substrates GalNAc-T activity -LA +LA Acceptor pmol/min/ng GlcNAc
31.3 3.3 Glc 2.6 34.8 Glucosamine 1.8 5.0 Assays were carries out
at 37.degree. C. using 500 .mu.M UDP-GalNAc for 10 minutes. In all
the measurements, the acceptor concentration was 100 mM and the LA
concentration was 55 .mu.m.
[0125] It is shown herein that the Tyr-289 mutants of Gal-T1
exhibit as high GalNAc-T activity as Gal-T activity, as well as
GlcNAc-T activity. In the human Gal-T family members, the Tyr/Phe
residue at 287 (or in bovine Tyr-289) is important for determining
the donor sugar specificity of the enzyme.
Example II
General Expression, Mutagenesis, Folding, and Purification of
Catalytic Domains of .beta.(1,4)-galactosyltransferase I Enzymes
Having an N-Terminal Stem Region
Materials and Methods
[0126] Restriction endonucleases and DNA-modifying enzymes were
from New England Biolabs (Beverly, Mass.). Oligonucleotide primers
were synthesized by the Recombinant DNA Facility at NCI-Frederick.
The plasmid mini preparation kit was from Qiagen (Santa Clarita,
Calif.). Ampicillin, UDP-Gal and UDP-agarose were from Sigma
Chemical Co, MO. pET23a vector and BL21 (.lamda.DE3)/pLysS
competent cells were from Novagen (Madison, Wis.). E. coli XL2-Blue
ultracompetent cells were from Stratagene (La Jolla, Calif.).
AG1-X8 resin, chloride form, 200-400 mesh was from Bio-Rad
(Hercules, Calif.). Taq DNA polymerase and PCR nucleotide mixes
were obtained from Boehringer Mannheim.
[0127] Cloning of Bovine and Human Stem-CD Clones of
.beta.1,4-galactosyltransferase:
[0128] Two sets of oligonucleotide primers with unique restriction
sites added at the 5' (SR) and 3' (CD) ends were synthesized based
upon the bovine .beta.4Gal-T1 and human .beta.4Gal-T1 cDNA sequence
(shown in FIGS. 3A and 3B). A full-length bovine cDNA clone
encoding .beta.4Gal-T1 was used for site-directed mutagenesis
(Boeggeman et al., Protein Eng. 6:779-785 (1993)). In the stem
region of bovine .beta.4Gal-T1 Ser96 was mutated to Ala96
(described below). The mutated full-length cDNA template was
amplified by the polymerase chain reaction (PCR), using the
following primer pair to generate SRCD.beta.4Gal-T1:
TABLE-US-00007 (SEQ ID NO: 17) (SR)
5'-CGCGGATCCCGCGACCTAAGACGCCTGCCTCAGCTGGTC-3' and (SEQ ID NO: 18)
(CD) 5'-TGGAATTCCTAGCTCGGCGTCCCGATGTCCACTGTGAT-3'.
Human placental QUICK-Clone cDNA (Lot #9020891) was obtained from
Clontech and used as the template for PCR. To amplify human
.beta.4Gal-T1 the following PCR primers were used:
TABLE-US-00008 (SEQ ID NO: 19) (SR)
5'-CGCGGATCCCGCGACCTGAGCCGCCTGCCCCAACTGGTC-3' and (SEQ ID NO: 20)
(CD) 5'-CCGGAATTCCTACTAGCTCGGTGTCCCGATGTCCACTGT- 3'.
The primer pairs amplified the cDNA region that has both the stem
and catalytic domain, and were designed to generate DNA fragments
containing a Bam HI (SR) and Eco RI (CD) (underlined) at the 5' and
3'-ends of the inserts, respectively. The PCR products were
purified using a QLAquick PCR purification kit and the purified
fragments were digested with Bam HI and Eco RI to generate the
cohesive ends. The expression constructs were obtained after
ligating the digested and gel-purified PCR fragments into the Bam
HI and Eco RI-digested pET23a vector (Novagen, Madison, Wis.) and
transformed into XL2-ultracompetent cells. Identities of the clones
were confirmed by DNA sequence.
[0129] Mutation of the stem region of bovine .beta.4Gal-T1 to
inhibit proteolysis: The wild-type bovine SRCD.beta.4Gal-T1, which
has Ser at position 96, was expressed in E. coli. Its sequence is
shown in FIG. 3A. The recombinant wild-type protein folded and
purified from inclusion bodies, as described below, was cleaved
within the SR region, over a short period of time. The cleaved
protein on SDS-PAGE shows a single band of molecular mass of 34.9
kDa. The protein was electroblotted to polyvinylidene difluoride
membrane, and the N-terminal sequence of the cleaved fragment was
determined by Edman degradation at the Protein Chemistry
Laboratory, NCI-Frederick. The N-terminal sequence of the fragment
was determined as SRAPSNLD (SEQ ID NO: 21). This corresponds to the
cleavage at Ser96 within the sequence region KPR.sup.96SRAPSNLD
(SEQ ID NO: 22). To prevent this proteolytic digestion within the
stem region, we now constructed a mutant substituting Ala in place
of Ser96. This prevented the cleavage within the stem region of
bovine .beta.4Gal-T1 keeping the recombinant protein intact for a
long period of time. Even though, the experimental results
described in this study are with the mutant bovine
S96A-SRCD.beta.4Gal-T1 (FIG. 3A), for the sake of convenience in
abbreviation this protein is simply referred to as bovine
SRCD.beta.4Gal-T1. The human SRCD.beta.4Gal-T1 also gets cleaved
within the stem region, at Val53 in the sequence QLV.sup.53GVSTPLQ
(SEQ ID NO: 23), but only after long periods of time. Since this
clone showed more stability than the bovine .beta.4Gal-T1, it was
not mutated and used as such for subsequent analysis.
[0130] Protein expression in E. coli and inclusion body isolation:
For protein expression BL21 (.lamda.DE3)/pLysS-competent cells were
transformed with the pET vector derivatives according to the
manufacturer's protocols. The transformed cells were grown in LB
broth containing 50 .mu.g.ml.sup.-1 ampicillin to an OD600 nm of
.about.0.7, followed by induction with 0.4 mM IPTG. Cultures were
harvested after 3-4 hours by centrifugation at 2000.times.g for 20
min. The inclusion bodies were isolated and solubilized as
described (Boeggeman et al., Protein Eng., 6:779-785 (1993)). From
a liter of induced bacterial culture, the yield is generally 80 to
100 mg of purified inclusion bodies. Novex gels were used for
SDS-PAGE analysis and the protein bands were visualized with
Coomassie blue. Protein concentrations were measured with the
Bio-Rad protein dye reagent with bovine serum albumin as the
standard.
[0131] In vitro folding of proteins from inclusion bodies: A
protocol utilizing oxido-shuffling agents within the renaturation
buffer was used to generate active recombinant .beta.4Gal-T1 from
the inclusion bodies (Boeggeman et al., Protein Eng., 6:779-785
(1993)). After renaturation and subsequent dialysis of the
solution, a portion of the folded protein precipitated in the form
of insoluble aggregates. To increase the yields of native proteins,
the inclusion bodies were sulfonated as previously described
(Boeggeman et al., Glycobiology 12:395-407 (2002)). The sulphonated
proteins were dissolved in 5 M guanidine-HCl, to a protein
concentration of 1 mg/ml, with an OD275 nm of 1.9 to 2.0. The
protein solution was then diluted 10-fold in small aliquots in a
renaturation solution to give a final protein concentration of 100
.mu.g/ml, in 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 0.5 M guanidine-HCl,
8 mM cysteamine and 4 mM cystamine. The protein was allowed to
renature for 48 hrs at 4.degree. C. and then dialyzed. The
precipitate was removed by centrifugation at 5000 rpm and the
supernatant concentrated using ultrafiltration membranes (Amicon,
Inc., Beverly, Mass., USA).
[0132] Improving the folding conditions: In recent years factorial
folding screens (Rudolph and Lilie, FASEB J., 10:40-56 (1996); Chen
and Gouaux, Proc. Natl. Acad. Sci. 94:13431-13436 (1997); Armstrong
et al., Prot. Sci., 8:1475-1483 (1999)) have been developed for
examining the folding efficiencies of proteins from inclusion
bodies. To improve the in vitro folding efficiency, 8 different
folding conditions similar to the formulations described in the
Foldlt Screen kit (Hampton Research, CA) with certain modifications
were tested. Condition I: 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 0.5 M
guanidine-HCl, 8 mM cysteamine and 4 mM cystamine. Condition II: 55
Mes pH 6.5, 10.56 mM NaCl, 0.44 mM KCl, 2.2 mM MgCl.sub.2, 2.2 mM
CaCl.sub.2, 0.5 M guanidine-HCl. Condition III: similar to
condition II with respect to the buffer, pH, chaotrope and salt
condition, but it had 0.055% PEG-4000, 1.1 mM EDTA, 0.44 M sucrose
and 0.55 M L-arginine. Condition IV: 55 mM Mes pH 6.5, 264 mM NaCl,
11 mM KCl, 0.055% PEG-4000, 0.5 M guanidine-HCl, 2.2 mM MgCl.sub.2,
2.2 mM CaCl.sub.2 and 0.44 M sucrose. Condition V: 55 mM Tris pH
8.2, 10.56 mM NaCl, 0.44 mM KCl, 1.1 mM EDTA, 0.44 M sucrose.
Conditions VI and VIII are similar except for the presence of redox
agents. Condition VII: 55 mM Mes pH 6.5, 264 mM NaCl, 11 mM KCl,
1.1 mM EDTA, 0.5 M guanidine-HCl, and 0.55 M L-arginine. The
buffers II through VII had 100 mM GSH and 10 mM GSSG. Conditions I
and VIII, had 8 mM cysteamine and 4 mM cystamine. Condition VIII,
gave the highest enzymatic activity, soluble and folded protein,
was 50 mM Tris-HCl pH 8.0, 10.56 mM NaCl, 0.44 mM KCl, 2.2 mM
MgCl.sub.2, 2.2 mM CaCl.sub.2 0.5 M guanidine-HCl, 8 mM cysteamine
and 4 mM cystamine, 0.055% PEG-4000 and 0.55 M L-arginine.
[0133] .beta.4Gal-T1 Enzyme Assays: The in vitro assay for
.beta.4Gal-T1 enzyme activity was performed as described (Boeggeman
et al., Glycobiology, 12:395-407 (2002)). The
.sup.3H-labeled-UDP-Gal was used as sugar donor and GlcNAc as the
sugar acceptor. A reaction without GlcNAc was used as a control.
The fusion SRCD.beta.4Gal-T1 proteins showed the same enzymatic
activities when compared to the wild-type recombinant .beta.4Gal-T1
(amount of product formation/min/mol of protein).
[0134] Binding of recombinant proteins to UDP-affinity column:
UDP-agarose was used to bind the active proteins at 4.degree. C.
Columns containing 1 ml bed volume of UDP-agarose were
pre-equilibrated with 10 ml of 25 mM cacodylic buffer, pH 7.6,
containing 25 mM MnCl.sub.2. The renatured protein solutions (0.5
mg) were adjusted to the pre-equilibration buffer conditions and
applied to the columns. After loading the sample, the pass-through
was re-cycled. It was then analyzed for the unbound protein by
SDS-PAGE. The columns were washed 3.times. with 1-ml portions of
equilibration buffer. The bound protein was eluted with the elution
buffer consisting of 25 mM cacodylic buffer, pH 7.6, 25 mM EDTA and
1 M NaCl. Fractions of 0.5 ml were collected from the columns, and
aliquots were analysed by SDS-PAGE. For enzymatic analysis the
appropriate fractions were pooled and dialysed against 50 mM
Tris-HCl pH 8.0 at 4.degree. C.
[0135] Circular dichroism-spectroscopy: Far ultraviolet circular
dichroism spectra of recombinant proteins was measured in a JASCO
J-715 spectropolarimeter. The spectra for each sample was scanned
at a speed of 100 nm/min. Far ultraviolet circular dichroism
spectra were obtained from 190 to 250 nm at room temperature using
a pathlength of 0.1 mm with a 1.0 nm bandwidth. Protein was
dissolved in Tris-HCl pH 8.0 at concentrations of 0.5 mg/ml. Buffer
blanks obtained in the same cuvette were subtracted to generate net
spectra. Raw ellipticity data were transformed into molar residue
ellipticity using the concentration values determined from optical
ultraviolet absorbance measurements. No smoothing or data averaging
was applied.
Results
Influence of the Stem Region (SR) Covalently Linked to the
N-Termini of the Catalytic Domain (CD) of the .beta.4Gal-T Family
Members on the In Vitro Folding Efficiency of the Recombinant
Proteins from Inclusion Bodies
[0136] The catalytic domain of .beta.4Gal-T1 (amino acid residues
130-402) (sequence in FIG. 3A, SEQ ID NO: 9) accumulated as an
insoluble material within the inclusion body fraction upon
expression in E. coli. Essentially all the proteins with the stem
regions covalently linked to the N-termini of the catalytic domain
(sequence in FIG. 3A, SEQ ID NO: 9) also accumulated in the
insoluble inclusion body fraction. Contaminating proteins from the
inclusion bodies were removed by washing the insoluble pellet
several times with a buffer containing 25% sucrose as described
herein. The yield of the purified inclusion bodies, varied with the
protein that is being expressed (Table VII). The proteins produced
a single protein band of expected molecular size on SDS-PAGE gel
(FIG. 4). These insoluble inclusion bodies required 5 M guanidine
HCl for solubilization.
[0137] Sulphonation of the inclusion bodies of CD.beta.4Gal-T1
prior to the in vitro folding step gave higher yields of final
folded proteins. The yield of sulphonated protein from the purified
inclusion bodies was generally half of the starting protein
material (Table VII). Oxido-shuffling reagents were present during
the renaturation step to regenerate the enzyme activity from all
the recombinant sulphonated proteins. The recombinant proteins
having the stem region fused at the N-termini of the catalytic
domain (SRCD) remained soluble during the renaturation step in 0.5
M guanidine HCl and the oxido-shuffling agents. Upon subsequent
dialysis of the SRCD-protein solutions, to remove the guanidine
HCl, both the folded and misfolded molecules remained soluble as
judged by SDS-PAGE analysis. In contrast, a majority of the
misfolded protein molecules aggregated and precipitated in the
protein solution containing only the CD. The recovery of soluble
human and bovine SRCD.beta.4Gal-T1 is 3- to 9-fold higher than
proteins having only the CD domain following refolding in the
absence of PEG-4000 and L-arginine. (Table VIII).
TABLE-US-00009 TABLE VII Yield of inclusion bodies and sulphonated
proteins Amount of sulphonated Recombinant Inclusion protein
recovered from Proteins body (mg)* inclusion bodies (mg).sup.+
Bovine CD.beta.4GalT-1 120 65 Bovine SRCD.beta.4GalT-1 113 50 Human
CD.beta.4GalT-1 80 37 Human SRCD.beta.4GalT-1 90 50 *Amount of
inclusion bodies obtained from the pellet of one liter of IPTG
induced bacterial culture. .sup.+Amount of sulphonated protein was
determined by SDS-PAGE and by protein estimation using the Bradford
method (Bio Rad).
TABLE-US-00010 TABLE VIII In vitro renaturation of the sulphonated
inclusion bodies -PEG/L-Arg.sup.+ +PEG/L-Arg.sup.+ CD.beta.4Gal-T1
SRCD.beta.4Gal-T1 CD.beta.4Gal-T1 SRCD.beta.4Gal-T1 mg
(%)*{circumflex over ( )} mg (%)*{circumflex over ( )} mg
(%)*{circumflex over ( )} mg (%)*{circumflex over ( )} Bovine 2.7
(14) 9.4 (47) 8.3 (42) 18.0 (90) Human 2.1 (11) 19.0 (95) 14.0 (69)
20.0 (100) .sup.+20 mg of sulphonated proteins were folded in the
buffer previously described that does not contain PEG-4000 and
L-arginine (condition I) (Boeggeman, et al.). The proteins after
the renaturation step were dialyzed, centrifuged and their
concentration determined by SDS-PAGE and the Bradford method
(BioRad) *Percentage recovery of the soluble protein after in vitro
folding of 20 mg of sulphonated .beta.4Gal-T1 {circumflex over (
)}The soluble renatured CD.beta.4Gal-T1 protein contains almost all
folded molecules, whereas the soluble renatured SRCD.beta.4Gal-T1
contains misfolded proteins that are removed on UDP-agarose columns
(see Table IX)
[0138] Renaturation conditions that increase the folding efficiency
and specific activity of .beta.4Gal-T1: After folding the bovine
wild-type CD.beta.4Gal-T1 in a renaturation buffer I (described
herein), the misfolded proteins precipitated out during subsequent
dialysis. In an attempt to increase the amount of the folded native
protein during the renaturation step, various folding conditions
consisting of seven different solutions were tested. The results
were compared to the yields obtained according to condition I (FIG.
5). The tested inclusion bodies were dissolved in 5 M guanidine HCl
to a final protein concentration of 1 mg/ml. Aliquots of 50 .mu.l
of the protein solution were added to 950 .mu.l of the folding
buffers in an eppendorf tube, mixed gently and incubated at
4.degree. C. for times ranging from 4 hours to overnight. The
folding buffers varied, (conditions I to VIII as disclosed herein)
with respect to: pH from 6.5 (in conditions II, III, IV and VII),
to 8.0 (in conditions V, VI, and VIII); cations (in conditions II,
IV, VI and VIII) and chelators (conditions I, III, V and VII).
Conditions VI, VII and VIII had polar additives (L-arginine).
Conditions IV and V had non-polar additives, such as sucrose.
Condition III had both sucrose and L-arginine. Some buffers also
had PEG4000 (conditions III, IV, VI and VIII). The appropriate
redox environment was created by the presence of 100 mM reduced
(GSH) and 10 mM oxidized (GSSH) glutathione, except under
conditions I and VIII which contained cysteamine (8 mM) and
cystamine (4 mM).
[0139] After overnight incubation and dialysis against 1 mM
Tris-HCl pH 8.0, samples were centrifuged and 5 .mu.l of
supernatant from each sample was tested for galactosyltransferase
activity and compared with each other (FIG. 5). The results showed
the highest activity under the buffer condition VI and VIII. Both
of these had 0.55 M L-arginine and 0.055% PEG-4000 (FIG. 5). All
folding conditions were tested in the presence of 30 mM lauryl
maltoside, which appears to have a negative effect on the folding
yields of the recombinant proteins, even in the presence of 0.55 M
L-arginine and 0.055% PEG-4060. The inclusion of sucrose had an
inhibitory effect (condition IV and V). No preference was observed
for pH, salts, or cations within the conditions tested. The redox
environment created by 8 mM cysteamine: 4 mM cystamine, (condition
VIII), was preferred over 100 mM GSH: 10 mM GSSH (condition VI).
Enzymatic assays (FIG. 5) revealed that the combination of
L-arginine and PEG-4000 in condition VIII, increased the in vitro
folding efficiency of the CD and SRCD proteins by about 7- and
3-fold, respectively, compared to condition I which did not contain
PEG4000 and L-arginine.
[0140] Misfolded proteins in the renatured SRCD.beta.Gal-T1:
Renatured proteins were analyzed on SDS-PAGE gels under native and
reducing conditions to determine whether the renatured
SRCD.beta.4Gal-T1 proteins were natively folded or misfolded.
Analyses under native conditions, in the absence of
.beta.-mercaptoethanol (.beta.ME) and without boiling the samples,
revealed that both bovine and human soluble SRCD.beta.4Gal-T1
proteins contained misfolded molecules that aggregated and remained
at the top of the gel (FIG. 6 (-)). Under reducing conditions,
using .beta.ME in the boiled samples, the misfolded molecules of
both bovine and human soluble SRCD.beta.4Gal-T1 proteins did not
appear on the top of the wells in SDS-PAGE and produced single
protein bands of the expected sizes (35 kDa for bovine species and
34 kDa for human species) (FIG. 6 (+)).
[0141] Binding of recombinant proteins to affinity columns: Folded
and active CD.beta.4Gal-T1 obtained from inclusion bodies bound to,
and was eluted from, a UDP-column with 25 mM EDTA and 1M NaCl.
Thus, binding of soluble renatured CD and SRCD.beta.4Gal-T1
proteins to UDP-agarose columns was tested. Native SDS gel analyses
of the soluble renatured CD.beta.4Gal-T1 protein, without .beta.ME
and boiling, showed that all of the soluble renatured CD protein
was natively folded. The renatured SRCD.beta.4Gal-T1 proteins were
then applied to a UDP-agarose column. Nearly all of the renatured
SRCD.beta.4Gal-T1 proteins bound to, and eluted from, the
UDP-agarose column (FIG. 7). The specific enzymatic activity of the
SRCD.beta.4Gal-T1 protein (product formation/min/ng protein) before
and after being passed through the UDP-agarose column was measured
to determine if the soluble protein fraction of the renatured
SRCD.beta.4Gal-T1 protein contained properly folded molecules (FIG.
8). Also, the samples were analyzed in the absence of .beta.ME and
without boiling. When condition I was used to fold bovine- and
human-SRCD proteins (FIG. 8A), the folded molecules that eluted
with 25 mM EDTA and 1 M NaCl, (FIG. 8A (+)) had 2- and 6-fold
higher specific activities (FIG. 8A, crossed-hatched bars),
respectively, when compared to the protein before loading onto the
affinity column (FIG. 8A, black bars). The renatured human
SRCD.beta.4Gal-T1 sample showed more misfolded proteins compared to
bovine SRCD.beta.4Gal-T1 (FIG. 8A, bovine versus human black bars).
In contrast, folding according to condition VIII, that contains
PEG-4000 and L-arginine (FIG. 8B), increased properly folded human
SRCD compared to condition I (FIG. 8B vs FIG. 8A, black bars, human
SRCD). Protein eluted from the UDP-column under condition VIII
(FIG. 8B, cross bar) had .about.2-fold higher specific activity as
compared to the proteins before being loaded on the affinity column
(FIG. 8B, black bars). Although soluble, a portion of the protein
in the SRCD samples is not folded to enable binding to UDP-agarose.
The increased specific activity of the eluted proteins indicated
the amount of misfolded molecules present in the sample before
being bound to the UDP-agarose column. These results agreed with
the observation that the misfolded proteins in the samples before
being passed through the UDP-agarose columns also remained at the
top of the wells in the SDS-PAGE gel (FIGS. 7B and C, lane U). The
soluble bovine and human CD.beta.4Gal-T1 behaved the same way
regardless of the buffer used for folding (condition I or VIII)
(Table IX). The specific activity of bovine and human
CD.beta.4Gal-T1 before and after binding on UDP-agarose columns did
not change.
TABLE-US-00011 TABLE IX Binding of in vitro folded .beta.4Gal-T1 to
UDP-agarose column -PEG/L-Arg +PEG/L-Arg CD.beta.4Gal-T1
SRCD.beta.4Gal-T1 CD.beta.4Gal-T1 SRCD.beta.4Gal-T1 % Input.sup.+
Output % Input.sup.+ Output Input.sup.+ Output % Input* Output %
yield* mg mg yield* mg mg mg mg yield* mg mg yield* Bovine 2.7 2.7
(14) 9.4 7.5 8.3 8.3 (42) 18.0 10.0 (50) (38) Human 2.1 2.1 (11)
19.0 12.2 14.0 14.0 (69) 20.0 11.0 (55) (61) .sup.+The amount of
soluble protein, recovered from 20 mg of sulphonated protein after
in vitro folding, loaded on UDP-agarose columns. *Percentage of
final protein yield from 20 mg of sulphonated inclusion bodies
after in vitro folding and passing through the UDP-agarose column.
All of the soluble folded CD.beta.4Gal-T1 proteins bind to and
elute from the UDP-agarose columns. On the other hand, the soluble
folded SRCD.beta.4Gal-T1 fraction contains misfolded proteins that
do not bind to the UDP-agarose column.
[0142] Circular dichroism-spectra of recombinant .beta.4Gal-T1:
Circular dichroism experiments were done with purified
.beta.4Gal-T1 proteins to determine if the addition of the stem
disturbed the overall secondary structure of .beta.4Gal-T1. No
significant differences in secondary structure relative to the
wild-type CD.beta.4Gal-T1 were detected for the bovine or human
proteins. All systems showed extrema of negative ellipticity
between 250 and 200 nm. The circular dichroism-spectrum of the
wild-type enzyme was highly reproducible and showed negative
extrema at 208 and 220 nm indicative of .alpha.-helicity. There was
a small change in ellipticity observed in the 200-210 nm region
upon the addition of the stem in both bovine and human SRCD
proteins.
[0143] All publications, patents and patent applications are
incorporated herein by reference. While in the foregoing
specification this invention has been described in relation to
certain preferred embodiments thereof, and many details have been
set forth for purposes of illustration, it will be apparent to
those skilled in the art that the invention is susceptible to
additional embodiments and that certain of the details described
herein may be varied considerably without departing from the basic
principles of the invention.
Sequence CWU 1
1
2317PRTHomo sapiens 1Phe Asn Arg Ala Lys Leu Leu 1 527PRTHomo
sapiens 2Tyr Val Gln Tyr Phe Gly Gly 1 53398PRTHomo sapiens 3Met
Arg Leu Arg Glu Pro Leu Leu Ser Arg Ser Ala Ala Met Pro Gly 1 5 10
15Ala Ser Leu Gln Arg Ala Cys Arg Leu Leu Val Ala Val Cys Ala Leu
20 25 30His Leu Gly Val Thr Leu Val Tyr Tyr Leu Ala Gly Arg Asp Leu
Ser 35 40 45Arg Leu Pro Gln Leu Val Gly Val Ser Thr Pro Leu Gln Gly
Gly Ser 50 55 60Asn Ser Ala Ala Ala Ile Gly Gln Ser Ser Gly Asp Leu
Arg Thr Gly65 70 75 80Gly Ala Arg Pro Pro Pro Pro Leu Gly Ala Ser
Ser Gln Pro Arg Pro 85 90 95Gly Gly Asp Ser Ser Pro Val Val Asp Ser
Gly Pro Gly Pro Ala Ser 100 105 110Asn Leu Thr Ser Val Pro Val Pro
His Thr Thr Ala Leu Ser Leu Pro 115 120 125Ala Cys Pro Glu Glu Ser
Pro Leu Leu Val Gly Pro Met Leu Ile Glu 130 135 140Phe Asn Met Pro
Val Asp Leu Glu Leu Val Ala Lys Gln Asn Pro Asn145 150 155 160Val
Lys Met Gly Gly Arg Tyr Ala Pro Arg Asp Cys Val Ser Pro His 165 170
175Lys Val Ala Ile Ile Ile Pro Phe Arg Asn Arg Gln Glu His Leu Lys
180 185 190Tyr Trp Leu Tyr Tyr Leu His Pro Val Leu Gln Arg Gln Gln
Leu Asp 195 200 205Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly Asp Thr
Ile Phe Asn Arg 210 215 220Ala Lys Leu Leu Asn Val Gly Phe Gln Glu
Ala Leu Lys Asp Tyr Asp225 230 235 240Tyr Thr Cys Phe Val Phe Ser
Asp Val Asp Leu Ile Pro Met Asn Asp 245 250 255His Asn Ala Tyr Arg
Cys Phe Ser Gln Pro Arg His Ile Ser Val Ala 260 265 270Met Asp Lys
Phe Gly Phe Ser Leu Pro Tyr Val Gln Tyr Phe Gly Gly 275 280 285Val
Ser Ala Ser Ser Lys Gln Gln Phe Leu Thr Ile Asn Gly Phe Pro 290 295
300Asn Asn Tyr Trp Gly Trp Gly Gly Glu Asp Asp Asp Ile Phe Asn
Arg305 310 315 320Leu Val Phe Arg Gly Met Ser Ile Ser Arg Pro Asn
Ala Val Val Gly 325 330 335Thr Cys Arg Met Ile Arg His Ser Arg Asp
Lys Lys Asn Glu Pro Asn 340 345 350Pro Gln Arg Phe Asp Arg Ile Ala
His Thr Lys Glu Thr Met Leu Ser 355 360 365Asp Gly Leu Asn Ser Leu
Thr Tyr Gln Val Leu Asp Val Gln Arg Tyr 370 375 380Pro Leu Tyr Thr
Gln Ile Thr Val Asp Ile Gly Thr Pro Ser385 390 3954399PRTMus
musculus 4Met Arg Phe Arg Glu Gln Phe Leu Gly Gly Ser Ala Ala Met
Pro Gly 1 5 10 15Ala Thr Leu Gln Arg Ala Cys Arg Leu Leu Val Ala
Val Cys Ala Leu 20 25 30His Leu Gly Val Thr Leu Val Tyr Tyr Leu Ser
Gly Arg Asp Leu Ser 35 40 45Arg Leu Pro Gln Leu Val Gly Val Ser Ser
Thr Leu Gln Gly Gly Thr 50 55 60Asn Gly Ala Ala Ala Ser Lys Gln Pro
Pro Gly Glu Gln Arg Pro Arg65 70 75 80Gly Ala Arg Pro Pro Pro Pro
Leu Gly Val Ser Pro Lys Pro Arg Pro 85 90 95Gly Leu Asp Ser Ser Pro
Gly Ala Ala Ser Gly Pro Gly Leu Lys Ser 100 105 110Asn Leu Ser Ser
Leu Pro Val Pro Thr Thr Thr Gly Leu Leu Ser Leu 115 120 125Pro Ala
Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met Leu Ile 130 135
140Asp Phe Asn Ile Ala Val Asp Leu Glu Leu Leu Ala Lys Lys Asn
Pro145 150 155 160Glu Ile Lys Thr Gly Gly Arg Tyr Ser Pro Lys Asp
Cys Val Ser Pro 165 170 175His Lys Val Ala Ile Ile Ile Pro Phe Arg
Asn Arg Gln Glu His Leu 180 185 190Lys Tyr Trp Leu Tyr Tyr Leu His
Pro Ile Leu Gln Arg Gln Gln Leu 195 200 205Asp Tyr Gly Ile Tyr Val
Ile Asn Gln Ala Gly Asp Thr Met Phe Asn 210 215 220Arg Ala Lys Leu
Leu Asn Ile Gly Phe Gln Glu Ala Leu Lys Asp Tyr225 230 235 240Asp
Tyr Asn Cys Phe Val Phe Ser Asp Val Asp Leu Ile Pro Met Asp 245 250
255Asp Arg Asn Ala Tyr Arg Cys Phe Ser Gln Pro Arg His Ile Ser Val
260 265 270Ala Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gln Tyr
Phe Gly 275 280 285Gly Val Ser Ala Leu Ser Lys Gln Gln Phe Leu Ala
Ile Asn Gly Phe 290 295 300Pro Asn Asn Tyr Trp Gly Trp Gly Gly Glu
Asp Asp Asp Ile Phe Asn305 310 315 320Arg Leu Val His Lys Gly Met
Ser Ile Ser Arg Pro Asn Ala Val Val 325 330 335Gly Arg Cys Arg Met
Ile Arg His Ser Arg Asp Lys Lys Asn Glu Pro 340 345 350Asn Pro Gln
Arg Phe Asp Arg Ile Ala His Thr Lys Glu Thr Met Arg 355 360 365Phe
Asp Gly Leu Asn Ser Leu Thr Tyr Lys Val Leu Asp Val Gln Arg 370 375
380Tyr Pro Leu Tyr Thr Gln Ile Thr Val Asp Ile Gly Thr Pro Arg385
390 3955402PRTBos taurus 5Met Lys Phe Arg Glu Pro Leu Leu Gly Gly
Ser Ala Ala Met Pro Gly 1 5 10 15Ala Ser Leu Gln Arg Ala Cys Arg
Leu Leu Val Ala Val Cys Ala Leu 20 25 30His Leu Gly Val Thr Leu Val
Tyr Tyr Leu Ala Gly Arg Asp Leu Arg 35 40 45Arg Leu Pro Gln Leu Val
Gly Val His Pro Pro Leu Gln Gly Ser Ser 50 55 60His Gly Ala Ala Ala
Ile Gly Gln Pro Ser Gly Glu Leu Arg Leu Arg65 70 75 80Gly Val Ala
Pro Pro Pro Pro Leu Gln Asn Ser Ser Lys Pro Arg Ser 85 90 95Arg Ala
Pro Ser Asn Leu Asp Ala Tyr Ser His Pro Gly Pro Gly Pro 100 105
110Gly Pro Gly Ser Asn Leu Thr Ser Ala Pro Val Pro Ser Thr Thr Thr
115 120 125Arg Ser Leu Thr Ala Cys Pro Glu Glu Ser Pro Leu Leu Val
Gly Pro 130 135 140Met Leu Ile Glu Phe Asn Ile Pro Val Asp Leu Lys
Leu Ile Glu Gln145 150 155 160Gln Asn Pro Lys Val Lys Leu Gly Gly
Arg Tyr Thr Pro Met Asp Cys 165 170 175Ile Ser Pro His Lys Val Ala
Ile Ile Ile Leu Phe Arg Asn Arg Gln 180 185 190Glu His Leu Lys Tyr
Trp Leu Tyr Tyr Leu His Pro Met Val Gln Arg 195 200 205Gln Gln Leu
Asp Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly Glu Ser 210 215 220Met
Phe Asn Arg Ala Lys Leu Leu Asn Val Gly Phe Lys Glu Ala Leu225 230
235 240Lys Asp Tyr Asp Tyr Asn Cys Phe Val Phe Ser Asp Val Asp Leu
Ile 245 250 255Pro Met Asn Asp His Asn Thr Tyr Arg Cys Phe Ser Gln
Pro Arg His 260 265 270Ile Ser Val Ala Met Asp Lys Phe Gly Phe Ser
Leu Pro Tyr Val Gln 275 280 285Tyr Phe Gly Gly Val Ser Ala Leu Ser
Lys Gln Gln Phe Leu Ser Ile 290 295 300Asn Gly Phe Pro Asn Asn Tyr
Trp Gly Trp Gly Gly Glu Asp Asp Asp305 310 315 320Ile Tyr Asn Arg
Leu Ala Phe Arg Gly Met Ser Val Ser Arg Pro Asn 325 330 335Ala Val
Ile Gly Lys Cys Arg Met Ile Arg His Ser Arg Asp Lys Lys 340 345
350Asn Glu Pro Asn Pro Gln Arg Phe Asp Arg Ile Ala His Thr Lys Glu
355 360 365Thr Met Leu Ser Asp Gly Leu Asn Ser Leu Thr Tyr Met Val
Leu Glu 370 375 380Val Gln Arg Tyr Pro Leu Tyr Thr Lys Ile Thr Val
Asp Ile Gly Thr385 390 395 400Pro Ser6113PRTHomo sapiens 6Arg Asp
Leu Ser Arg Leu Pro Gln Leu Val Gly Val Ser Thr Pro Leu 1 5 10
15Gln Gly Gly Ser Asn Ser Ala Ala Ala Ile Gly Gln Ser Ser Gly Asp
20 25 30Leu Arg Thr Gly Gly Ala Arg Pro Pro Pro Pro Leu Gly Ala Ser
Ser 35 40 45Gln Pro Arg Pro Gly Gly Asp Ser Ser Pro Val Val Asp Ser
Gly Pro 50 55 60Gly Pro Ala Ser Asn Leu Thr Ser Val Pro Val Pro His
Thr Thr Ala65 70 75 80Leu Ser Leu Pro Ala Cys Pro Glu Glu Ser Pro
Leu Leu Val Gly Pro 85 90 95Met Leu Ile Glu Phe Asn Met Pro Val Asp
Leu Glu Leu Val Ala Lys 100 105 110Gln785PRTBos taurus 7Arg Asp Leu
Arg Arg Leu Pro Gln Leu Val Gly Val His Pro Pro Leu 1 5 10 15Gln
Gly Ser Ser His Gly Ala Ala Ala Ile Gly Gln Pro Ser Gly Glu 20 25
30Leu Arg Leu Arg Gly Val Ala Pro Pro Pro Pro Leu Gln Asn Ser Ser
35 40 45Lys Pro Arg Ser Arg Ala Pro Ser Asn Leu Asp Ala Tyr Ser His
Pro 50 55 60Gly Pro Gly Pro Gly Pro Gly Ser Asn Leu Thr Ser Ala Pro
Val Pro65 70 75 80Ser Thr Thr Thr Arg 858273PRTHomo sapiens 8Ser
Leu Pro Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met 1 5 10
15Leu Ile Glu Phe Asn Met Pro Val Asp Leu Glu Leu Val Ala Lys Gln
20 25 30Asn Pro Asn Val Lys Met Gly Gly Arg Tyr Ala Pro Arg Asp Cys
Val 35 40 45Ser Pro His Lys Val Ala Ile Ile Ile Pro Phe Arg Asn Arg
Gln Glu 50 55 60His Leu Lys Tyr Trp Leu Tyr Tyr Leu His Pro Val Leu
Gln Arg Gln65 70 75 80Gln Leu Asp Tyr Gly Ile Tyr Val Ile Asn Gln
Ala Gly Asp Thr Ile 85 90 95Phe Asn Arg Ala Lys Leu Leu Asn Val Gly
Phe Gln Glu Ala Leu Lys 100 105 110Asp Tyr Asp Tyr Thr Cys Phe Val
Phe Ser Asp Val Asp Leu Ile Pro 115 120 125Met Asn Asp His Asn Ala
Tyr Arg Cys Phe Ser Gln Pro Arg His Ile 130 135 140Ser Val Ala Met
Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gln Tyr145 150 155 160Phe
Gly Gly Val Ser Ala Ser Ser Lys Gln Gln Phe Leu Thr Ile Asn 165 170
175Gly Phe Pro Asn Asn Tyr Trp Gly Trp Gly Gly Glu Asp Asp Asp Ile
180 185 190Phe Asn Arg Leu Val Phe Arg Gly Met Ser Ile Ser Arg Pro
Asn Ala 195 200 205Val Val Gly Thr Cys Arg Met Ile Arg His Ser Arg
Asp Lys Lys Asn 210 215 220Glu Pro Asn Pro Gln Arg Phe Asp Arg Ile
Ala His Thr Lys Glu Thr225 230 235 240Met Leu Ser Asp Gly Leu Asn
Ser Leu Thr Tyr Gln Val Leu Asp Val 245 250 255Gln Arg Tyr Pro Leu
Tyr Thr Gln Ile Thr Val Asp Ile Gly Thr Pro 260 265
270Ser9273PRTBos taurus 9Ser Leu Thr Ala Cys Pro Glu Glu Ser Pro
Leu Leu Val Gly Pro Met 1 5 10 15Leu Ile Glu Phe Asn Ile Pro Val
Asp Leu Lys Leu Ile Glu Gln Gln 20 25 30Asn Pro Lys Val Lys Leu Gly
Gly Arg Tyr Thr Pro Met Asp Cys Ile 35 40 45Ser Pro His Lys Val Ala
Ile Ile Ile Leu Phe Arg Asn Arg Gln Glu 50 55 60His Leu Lys Tyr Trp
Leu Tyr Tyr Leu His Pro Met Val Gln Arg Gln65 70 75 80Gln Leu Asp
Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly Glu Ser Met 85 90 95Phe Asn
Arg Ala Lys Leu Leu Asn Val Gly Phe Lys Glu Ala Leu Lys 100 105
110Asp Tyr Asp Tyr Asn Cys Phe Val Phe Ser Asp Val Asp Leu Ile Pro
115 120 125Met Asn Asp His Asn Thr Tyr Arg Cys Phe Ser Gln Pro Arg
His Ile 130 135 140Ser Val Ala Met Asp Lys Phe Gly Phe Ser Leu Pro
Tyr Val Gln Tyr145 150 155 160Phe Gly Gly Val Ser Ala Leu Ser Lys
Gln Gln Phe Leu Ser Ile Asn 165 170 175Gly Phe Pro Asn Asn Tyr Trp
Gly Trp Gly Gly Glu Asp Asp Asp Ile 180 185 190Tyr Asn Arg Leu Ala
Phe Arg Gly Met Ser Val Ser Arg Pro Asn Ala 195 200 205Val Ile Gly
Lys Cys Arg Met Ile Arg His Ser Arg Asp Lys Lys Asn 210 215 220Glu
Pro Asn Pro Gln Arg Phe Asp Arg Ile Ala His Thr Lys Glu Thr225 230
235 240Met Leu Ser Asp Gly Leu Asn Ser Leu Thr Tyr Met Val Leu Glu
Val 245 250 255Gln Arg Tyr Pro Leu Tyr Thr Lys Ile Thr Val Asp Ile
Gly Thr Pro 260 265 270Ser101197PRTHomo sapiens 10Ala Thr Gly Ala
Gly Gly Cys Thr Thr Cys Gly Gly Gly Ala Gly Cys 1 5 10 15Cys Gly
Cys Thr Cys Cys Thr Gly Ala Gly Cys Cys Gly Gly Ala Gly 20 25 30Cys
Gly Cys Cys Gly Cys Gly Ala Thr Gly Cys Cys Ala Gly Gly Cys 35 40
45Gly Cys Gly Thr Cys Cys Cys Thr Ala Cys Ala Gly Cys Gly Gly Gly
50 55 60Cys Cys Thr Gly Cys Cys Gly Cys Cys Thr Gly Cys Thr Cys Gly
Thr65 70 75 80Gly Gly Cys Cys Gly Thr Cys Thr Gly Cys Gly Cys Thr
Cys Thr Gly 85 90 95Cys Ala Cys Cys Thr Thr Gly Gly Cys Gly Thr Cys
Ala Cys Cys Cys 100 105 110Thr Cys Gly Thr Thr Thr Ala Cys Thr Ala
Cys Cys Thr Gly Gly Cys 115 120 125Thr Gly Gly Cys Cys Gly Cys Gly
Ala Cys Cys Thr Gly Ala Gly Cys 130 135 140Cys Gly Cys Cys Thr Gly
Cys Cys Cys Cys Ala Ala Cys Thr Gly Gly145 150 155 160Thr Cys Gly
Gly Ala Gly Thr Cys Thr Cys Cys Ala Cys Ala Cys Cys 165 170 175Gly
Cys Thr Gly Cys Ala Gly Gly Gly Cys Gly Gly Gly Thr Cys Gly 180 185
190Ala Ala Cys Ala Gly Thr Gly Cys Cys Gly Cys Cys Gly Cys Cys Ala
195 200 205Thr Cys Gly Gly Gly Cys Ala Gly Thr Cys Cys Thr Cys Cys
Gly Gly 210 215 220Gly Gly Ala Cys Cys Thr Cys Cys Gly Gly Ala Cys
Cys Gly Gly Ala225 230 235 240Gly Gly Gly Gly Cys Cys Cys Gly Gly
Cys Cys Gly Cys Cys Gly Cys 245 250 255Cys Thr Cys Cys Thr Cys Thr
Ala Gly Gly Cys Gly Cys Cys Thr Cys 260 265 270Cys Thr Cys Cys Cys
Ala Gly Cys Cys Gly Cys Gly Cys Cys Cys Gly 275 280 285Gly Gly Thr
Gly Gly Cys Gly Ala Cys Thr Cys Cys Ala Gly Cys Cys 290 295 300Cys
Ala Gly Thr Cys Gly Thr Gly Gly Ala Thr Thr Cys Thr Gly Gly305 310
315 320Cys Cys Cys Thr Gly Gly Cys Cys Cys Cys Gly Cys Thr Ala Gly
Cys 325 330 335Ala Ala Cys Thr Thr Gly Ala Cys Cys Thr Cys Gly Gly
Thr Cys Cys 340 345 350Cys Ala Gly Thr Gly Cys Cys Cys Cys Ala Cys
Ala Cys Cys Ala Cys 355 360 365Cys Gly Cys Ala Cys Thr Gly Thr Cys
Gly Cys Thr Gly Cys Cys Cys 370 375 380Gly Cys Cys Thr Gly Cys Cys
Cys Thr Gly Ala Gly Gly Ala Gly Thr385 390 395 400Cys Cys Cys Cys
Gly Cys Thr Gly Cys Thr Thr Gly Thr Gly Gly Gly 405 410 415Cys Cys
Cys Cys Ala Thr Gly Cys Thr Gly Ala Thr Thr Gly Ala Gly 420 425
430Thr Thr Thr Ala Ala Cys Ala Thr Gly Cys Cys Thr Gly Thr Gly Gly
435 440 445Ala Cys Cys Thr Gly Gly Ala Gly Cys Thr Cys Gly Thr Gly
Gly Cys 450 455 460Ala Ala Ala Gly Cys Ala Gly Ala Ala Cys Cys Cys
Ala Ala Ala Thr465 470 475 480Gly Thr Gly Ala Ala Gly Ala Thr Gly
Gly Gly Cys Gly Gly Cys Cys 485 490 495Gly Cys Thr Ala Thr
Gly Cys Cys Cys Cys Cys Ala Gly Gly Gly Ala 500 505 510Cys Thr Gly
Cys Gly Thr Cys Thr Cys Thr Cys Cys Thr Cys Ala Cys 515 520 525Ala
Ala Gly Gly Thr Gly Gly Cys Cys Ala Thr Cys Ala Thr Cys Ala 530 535
540Thr Thr Cys Cys Ala Thr Thr Cys Cys Gly Cys Ala Ala Cys Cys
Gly545 550 555 560Gly Cys Ala Gly Gly Ala Gly Cys Ala Cys Cys Thr
Cys Ala Ala Gly 565 570 575Thr Ala Cys Thr Gly Gly Cys Thr Ala Thr
Ala Thr Thr Ala Thr Thr 580 585 590Thr Gly Cys Ala Cys Cys Cys Ala
Gly Thr Cys Cys Thr Gly Cys Ala 595 600 605Gly Cys Gly Cys Cys Ala
Gly Cys Ala Gly Cys Thr Gly Gly Ala Cys 610 615 620Thr Ala Thr Gly
Gly Cys Ala Thr Cys Thr Ala Thr Gly Thr Thr Ala625 630 635 640Thr
Cys Ala Ala Cys Cys Ala Gly Gly Cys Gly Gly Gly Ala Gly Ala 645 650
655Cys Ala Cys Thr Ala Thr Ala Thr Thr Cys Ala Ala Thr Cys Gly Thr
660 665 670Gly Cys Thr Ala Ala Gly Cys Thr Cys Cys Thr Cys Ala Ala
Thr Gly 675 680 685Thr Thr Gly Gly Cys Thr Thr Thr Cys Ala Ala Gly
Ala Ala Gly Cys 690 695 700Cys Thr Thr Gly Ala Ala Gly Gly Ala Cys
Thr Ala Thr Gly Ala Cys705 710 715 720Thr Ala Cys Ala Cys Cys Thr
Gly Cys Thr Thr Thr Gly Thr Gly Thr 725 730 735Thr Thr Ala Gly Thr
Gly Ala Cys Gly Thr Gly Gly Ala Cys Cys Thr 740 745 750Cys Ala Thr
Thr Cys Cys Ala Ala Thr Gly Ala Ala Thr Gly Ala Thr 755 760 765Cys
Ala Thr Ala Ala Thr Gly Cys Gly Thr Ala Cys Ala Gly Gly Thr 770 775
780Gly Thr Thr Thr Thr Thr Cys Ala Cys Ala Gly Cys Cys Ala Cys
Gly785 790 795 800Gly Cys Ala Cys Ala Thr Thr Thr Cys Cys Gly Thr
Thr Gly Cys Ala 805 810 815Ala Thr Gly Gly Ala Thr Ala Ala Gly Thr
Thr Thr Gly Gly Ala Thr 820 825 830Thr Cys Ala Gly Cys Cys Thr Ala
Cys Cys Thr Thr Ala Thr Gly Thr 835 840 845Thr Cys Ala Gly Thr Ala
Thr Thr Thr Thr Gly Gly Ala Gly Gly Thr 850 855 860Gly Thr Cys Thr
Cys Thr Gly Cys Thr Thr Cys Ala Ala Gly Thr Ala865 870 875 880Ala
Ala Cys Ala Ala Cys Ala Gly Thr Thr Thr Cys Thr Ala Ala Cys 885 890
895Cys Ala Thr Cys Ala Ala Thr Gly Gly Ala Thr Thr Thr Cys Cys Thr
900 905 910Ala Ala Thr Ala Ala Thr Thr Ala Thr Thr Gly Gly Gly Gly
Cys Thr 915 920 925Gly Gly Gly Gly Ala Gly Gly Ala Gly Ala Ala Gly
Ala Thr Gly Ala 930 935 940Thr Gly Ala Cys Ala Thr Thr Thr Thr Thr
Ala Ala Cys Ala Gly Ala945 950 955 960Thr Thr Ala Gly Thr Thr Thr
Thr Thr Ala Gly Ala Gly Gly Cys Ala 965 970 975Thr Gly Thr Cys Thr
Ala Thr Ala Thr Cys Thr Cys Gly Cys Cys Cys 980 985 990Ala Ala Ala
Thr Gly Cys Thr Gly Thr Gly Gly Thr Cys Gly Gly Gly 995 1000
1005Ala Cys Gly Thr Gly Thr Cys Gly Cys Ala Thr Gly Ala Thr Cys Cys
1010 1015 1020Gly Cys Cys Ala Cys Thr Cys Ala Ala Gly Ala Gly Ala
Cys Ala Ala1025 1030 1035 1040Gly Ala Ala Ala Ala Ala Thr Gly Ala
Ala Cys Cys Cys Ala Ala Thr 1045 1050 1055Cys Cys Thr Cys Ala Gly
Ala Gly Gly Thr Thr Thr Gly Ala Cys Cys 1060 1065 1070Gly Ala Ala
Thr Thr Gly Cys Ala Cys Ala Cys Ala Cys Ala Ala Ala 1075 1080
1085Gly Gly Ala Gly Ala Cys Ala Ala Thr Gly Cys Thr Cys Thr Cys Thr
1090 1095 1100Gly Ala Thr Gly Gly Thr Thr Thr Gly Ala Ala Cys Thr
Cys Ala Cys1105 1110 1115 1120Thr Cys Ala Cys Cys Thr Ala Cys Cys
Ala Gly Gly Thr Gly Cys Thr 1125 1130 1135Gly Gly Ala Thr Gly Thr
Ala Cys Ala Gly Ala Gly Ala Thr Ala Cys 1140 1145 1150Cys Cys Ala
Thr Thr Gly Thr Ala Thr Ala Cys Cys Cys Ala Ala Ala 1155 1160
1165Thr Cys Ala Cys Ala Gly Thr Gly Gly Ala Cys Ala Thr Cys Gly Gly
1170 1175 1180Gly Ala Cys Ala Cys Cys Gly Ala Gly Cys Thr Ala
Gly1185 1190 11951142DNAArtificial SequenceA synthetic primer
11ccttacgtgc aattgtttgg aggtgtctct gctctaagta aa
421236DNAArtificial SequenceA synthetic primer 12gacacctcca
aacaattgca cgtaaggtag gctaaa 361341DNAArtificial SequenceA
synthetic primer 13ctaccttacg tgcagatctt tggaggtgtc tctgctctaa g
411440DNAArtificial SequenceA synthetic primer 14gacacctcca
aagatctgca cgtaaggtag gctaatccaa 401541DNAArtificial SequenceA
synthetic primer 15ggattagcct accatatgtg cagaattttg gaggtgtctc t
411642DNAArtificial SequenceA synthetic primer 16agagacacct
ccaaaattct gcacatctgg taggctaaat cc 421739DNAArtificial SequenceA
synthetic primer 17cgcggatccc gcgacctaag acgcctgcct cagctggtc
391838DNAArtificial SequenceA synthetic primer 18tggaattcct
agctcggcgt cccgatgtcc actgtgat 381939DNAHomo sapiens 19cgcggatccc
gcgacctgag ccgcctgccc caactggtc 392039DNAHomo sapiens 20ccggaattcc
tactagctcg gtgtcccgat gtccactgt 39218PRTBos taurus 21Ser Arg Ala
Pro Ser Asn Leu Asp 1 52211PRTBos taurus 22Lys Pro Arg Ser Arg Ala
Pro Ser Asn Leu Asp 1 5 102310PRTHomo sapiens 23Gln Leu Val Gly Val
Ser Thr Pro Leu Gln 1 5 10
* * * * *