U.S. patent application number 12/040141 was filed with the patent office on 2009-01-08 for human spasmolytic polypeptide in glycosylated form.
This patent application is currently assigned to Novo Nordisk A/S. Invention is credited to Soren Erik Bjorn, Mogens Christensen, Per Franklin Nielsen, Fanny Norris, Kjeld Norris, Lars Thim.
Application Number | 20090011461 12/040141 |
Document ID | / |
Family ID | 8089375 |
Filed Date | 2009-01-08 |
United States Patent
Application |
20090011461 |
Kind Code |
A1 |
Thim; Lars ; et al. |
January 8, 2009 |
Human Spasmolytic Polypeptide in Glycosylated Form
Abstract
Human spasmolytic polypeptide (HSP) which has the amino acid
sequence Glu Lys Pro Ser Pro Cys Gln Cys Ser Arg Leu Ser Pro His
Asn Arg Thr Asn Cys Gly Phe Pro Gly Ile Thr Ser Asp Gln Cys Phe Asp
Asn Gly Cys Cys Phe Asp Ser Ser Val Thr Gly Val Pro Trp Cys Phe His
Pro Leu Pro Lys Gln Glu Ser Asp Gln Cys Val Met Glu Val Ser Asp Arg
Arg Asn Cys Gly Tyr Pro Gly Ile Ser Pro Glu Glu Cys Ala Ser Arg Lys
Cys Cys Phe Ser Asn Phe Ile Phe Glu Val Pro Trp Cys Phe Phe Pro Asn
Ser Val Glu Asp Cys His Tyr or a functionally equivalent homologue
thereof, characterized by being in glycosylated form.
Inventors: |
Thim; Lars; (Gentofte,
DK) ; Norris; Kjeld; (Hellerup, DK) ; Norris;
Fanny; (Hellerup, DK) ; Bjorn; Soren Erik;
(Lyngby, DK) ; Christensen; Mogens; (Gentofte,
DK) ; Nielsen; Per Franklin; (Vaerlose, DK) |
Correspondence
Address: |
NOVO NORDISK, INC.;INTELLECTUAL PROPERTY DEPARTMENT
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
|
Assignee: |
Novo Nordisk A/S
Bagsvaerd
DK
|
Family ID: |
8089375 |
Appl. No.: |
12/040141 |
Filed: |
February 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11139749 |
May 27, 2005 |
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12040141 |
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09528644 |
Mar 20, 2000 |
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11139749 |
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09027893 |
Feb 23, 1998 |
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09528644 |
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08491976 |
Aug 2, 1995 |
5783416 |
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09027893 |
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PCT/DK94/00037 |
Jan 20, 1994 |
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08491976 |
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Current U.S.
Class: |
435/69.1 |
Current CPC
Class: |
C07K 14/575 20130101;
A61P 43/00 20180101; A61K 38/00 20130101 |
Class at
Publication: |
435/69.1 |
International
Class: |
C12P 21/04 20060101
C12P021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 21, 1993 |
DK |
0068/93 |
Claims
1. A method of preparing a glycosylated human spasmolytic
polypeptide (hSP) having an amino acid sequence according to SEQ ID
NO 1, said method comprising: (a) culturing a yeast cell containing
a DNA sequence encoding said hSP under conditions permitting
production and secretion of said hSP from the yeast cell; and (b)
recovering the resulting secreted hSP, wherein the recovered hSP is
glycosylated at Asn 15 and is useful in the treatment of peptic
ulcers.
2. The method of claim 1, wherein the yeast cell is selected from
the group consisting of Saccharomyces cerevisiae, Saccharomyces
kluyveri, Schizosaccharomyces pombe or Saccharomyces uvarum.
3. The method of claim 2, wherein the yeast cell is Saccharomyces
cerevisiae.
4. The method of claim 1, wherein the recovered hSP is glycosylated
at Asn 15 with (GlcNAc).sub.2(Man).sub.10-15.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/139,749, filed May 27, 2005, which is a divisional of U.S.
application Ser. No. 09/528,644 filed 20 Mar. 2000, which is a
divisional of U.S. application Ser. No. 09/027,893 filed Feb. 23,
1998 which is a continuation of U.S. application Ser. No.
08/491,976 filed Aug. 2, 1995, now U.S. Pat. No. 5,783,416, which
is a continuation of PCT/DK94/00037 filed Jan. 20, 1994, and claims
priority under 35 U.S.C. 119 of Danish application serial no.
0068/93 filed Jan. 21, 1993, which is incorporated herein by
reference.
FIELD OF INVENTION
[0002] The present invention relates to human spasmolytic
polypeptide in glycosylated form, variants of human and porcine
spasmolytic polypeptides and a method of producing spasmolytic
polypeptides in glycosylated form.
BACKGROUND OF THE INVENTION
[0003] Human spasmolytic polypeptide (HSP) belongs to a family of
peptides containing one or more characteristic trefoil domains [1].
The trefoil domain is made up of a sequence of 38 or 39 amino acid
residues in which 6 cystein residues are linked in the
configuration 1-5, 2-4 and 3-6 thus forming a characteristic
trefoil structure [1]. The trefoil family of peptides consists of
rat intestinal trefoil factor, ITF [2], human breast cancer
associated peptide, pS2 [3, 4, 5], porcine, human and murine
spasmolytic polypeptide (PSP, HSP, MSP) [6, 7, 8] and frog
spasmolysins (xP1, xP2 and xP4) [8, 10, 11] all containing 1, 2 or
4 trefoil domains (FIG. 1).
[0004] The physiological function of the trefoil peptides is poorly
understood, and so far only PSP has been studied in any detail. In
the porcine pancreas, PSP is found in the acinar cells and to be
secreted in large amounts (50-100 mg/ml) into the pancreatic juice
upon stimulation with pancreozymin or secretin [12, 13, 14]. PSP is
resistant to digestion by intestinal proteases in the
gastrointestinal tract [12], and specific binding of PSP to rat
intestinal mucosa cells and membrane preparations from these cells
has been demonstrated [15, 16]. In the porcine gastrointestinal
tract, specific receptor-like binding to Paneth cells in the
duodenum has been found [17]. These results suggest a unique
intraluminal function of the peptide. A pharmacological screening
has indicated that PSP has spasmolytic and gastric acid secretion
inhibitory effects [18], and studies on mammalian cells have
indicated a growth factor-like activity of PSP [19].
[0005] The DNA sequence and derived amino acid sequence of the
human counterpart of porcine SP is shown in [8]. Unlike PSP, human
SP (FIG. 2), has been found to be expressed in the stomach, but not
in the pancreas to any greater extent [8]. An increased expression
of HSP and pS2 has been reported to be associated with peptic
ulcers and mucosal injury in inflammatory bowel disease [20, 21]
indicating a possible healing function of these peptides.
[0006] Only very limited amounts of HSP can be prepared by
extraction of human tissue. An object of study resulting in the
present invention was therefore to prepare recombinant HSP in
sufficient amounts for physiological and biochemical studies of the
peptide.
SUMMARY OF THE INVENTION
[0007] It has surprisingly been found that when recombinant HSP is
produced in certain host organisms, a proportion of it is produced
in glycosylated form by posttranslational modifications. The
glycosylated form of HSP has not, to applicant's best knowledge,
been described previously.
[0008] Accordingly, the present invention relates to human
spasmolytic polypeptide (HSP) which has the amino acid sequence
TABLE-US-00001 (SEQ ID NO:1) Glu Lys Pro Ser Pro Cys Gln Cys Ser
Arg Leu Ser Pro His Asn Arg Thr Asn Cys Gly Phe Pro Gly Ile Thr Ser
Asp Gln Cys Phe Asp Asn Gly Cys Cys Phe Asp Ser Ser Val Thr Gly Val
Pro Trp Cys Phe His Pro Leu Pro Lys Gln Glu Ser Asp Gln Cys Val Met
Glu Val Ser Asp Arg Arg Asn Cys Gly Tyr Pro Gly Ile Ser Pro Glu Glu
Cys Ala Ser Arg Lys Cys Cys Phe Ser Asn Phe Ile Phe Glu Val Pro Trp
Cys Phe Phe Pro Asn Ser Val Glu Asp Cys His Tyr
or a functionally equivalent homologue thereof, characterized by
being in glycosylated form.
[0009] In the present context, the term "functionally equivalent"
is intended to indicate that the homologous polypeptide has a
biological activity (e.g. spasmolytic effect) corresponding to that
of native HSP. The term "homologue" is intended to indicate a
polypeptide encoded by DNA which hybridizes to the same probe as
the DNA coding for HSP under comditions of high or low stringency
(e.g. as described in Sambrook et. al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989). More specifically, the term is
intended to refer to a DNA sequence which is at least 60%
homologous to the sequence encoding HSP with the amino acid
sequence shown above. The term is intended to include modifications
of the DNA sequence such as nucleotide substitutions which do not
give rise to another amino acid sequence of the polypeptide, but
which correspond to the codon usage of the host organism into which
the DNA construct is introduced or nucleotide substitutions which
do give rise to a different amino acid sequence and therefore,
possibly, a different protein structure which might give rise to a
mutant polypeptide with different properties than the native
enzyme. Other examples of possible modifications are insertion of
one or more codons into the sequence, addition of one or more
codons at either end of the sequence, or deletion of one or more
codons at either end or within the sequence. The term
"glycosylated" is intended to indicate that a carbohydrate moiety
is present at one or more sites of the protein molecule. It is at
present contemplated that glycosylation of HSP may give rise to
differences in the biological activity of the protein, for instance
with respect to stability towards proteolytic enzymes in the
gastrointestinal tract, solubility at gastric and/or intestinal pH
compared to non-glycosylated HSP, antigenicity, half-life, tertiary
structure, and targeting to receptors on appropriate cells.
[0010] In another aspect, the present invention relates to a
variant of a spasmolytic polypeptide (SP) which is a fragment of
human spasmolytic polypeptide (HSP) or porcine spasmolytic
polypeptide (PSP) comprising at least one trefoil domain.
[0011] The variant SP may be provided in both glycosylated and
non-glycosylated form. It is at present contemplated that such a
variant may be advantageous to use instead of full-length SP
because of a higher specific biological activity, increased
solubility and stability, longer half-life, easier way of
production, or the like.
[0012] It is assumed that other spasmolytic polypeptides than HSP
will, if provided with a glycosylation site, also be expressed in
predominantly glycosylated form. In a further aspect, the present
invention therefore relates to a method of preparing a spasmolytic
polypeptide in at least 60% glycosylated form, wherein a host cell
transformed with a DNA fragment encoding a spasmolytic polypeptide
and capable of providing glycosylation of said spasmolytic
polypeptide is cultured under conditions permitting production of
said spasmolytic polypeptide and recovering the resulting
spasmolytic polypeptide from the culture.
DETAILED DESCRIPTION OF THE INVENTION
[0013] It has been found that, at least when recombinant HSP is
produced in yeast, the proportion of it that is provided in
glycosylated form is in N-glycosylated form. It has further been
found that glycosylation takes place at Asn15 of the sequence shown
above. In preferred embodiments of glycosylated HSP, the
glycosylated side chain contains at least one hexose unit. In
particular, the glycosylated side chain may contain at least one
mannose unit, preferably at least five mannose units, most
preferably at least ten mannose units. In one preferred embodiment
of glycosylated HSP of the invention, the glycosylated side chain
contains 13-17 mannose units. In other preferred embodiments, the
glycosylated HSP is in addition glycosylated with at least one unit
of N-acetyl glucosamine (GlcNAc). In the currently preferred
embodiment, the glycosylated HSP is glycosylated at Asn15 with
(GlcNAc).sub.2(Man).sub.10-15.
[0014] It is further contemplated to produce homologues of HSP
which are provided with one or more additional glycosylation sites.
Thus, the present invention also relates to HSP homologues, wherein
Lys2 is replaced by Asn, Gln7 is replaced by Asn, Arg10 is replaced
by Asn, Gly 20 is replaced by Thr or Ser, Gly23 is replaced by Asn,
Ile 24 is replaced by Asn, Phe 36 is replaced by Asn, Asp 37 is
replaced by Asn, Ser39 is replaced by Asn, Gln53 is replaced by
Asn, Glu61 is replaced by Asn, Asp64 is replaced by Asn, Arg66 is
replaced by Thr or Ser, Gly69 is replaced by Thr or Ser, Gly72 is
replaced by Asn, Ile 89 is replaced by Thr or Ser, Pro98 is
replaced by Asn or Val101 is replaced by Thr or Ser, or a
combination of two or more of these substitutions. In a currently
preferred embodiment of such an HSP homologue, Asp64 is replaced by
Asn, and Arg66 is replaced by Thr or Ser.
[0015] It is of course understood that HSP homologues of the
invention may be glycosylated in the same manner at one or more of
these sites as described above for glycosylation at Asn15.
[0016] It is assumed that the trefoil structure common among
spasmolytic polypeptides is important for the function of HSP and
PSP. The variant human or porcine SP comprising a fragment of the
full-length polypeptide should therefore include at least three
disulfide bonds to provide this structure. Consequently, the
variant may comprise at least a sequence of amino acids from
position 8 to 46 or from position 58 to 95, each of which sequences
defines a trefoil domain of HSP and PSP.
[0017] As indicated above, the SP variant of the invention may be
provided in non-glycosylated form. This may, for instance, be
accomplished by substituting Asn15 by another amino acid, e.g. Asp
or Glu, or by substituting Thr17 by another amino acid except Ser,
e.g. Ala. It is more likely, however, that one or more additional
glycosylation sites will be introduced into this domain, for
instance by replacing Arg10 by Asn, Gly 20 by Thr or Ser, Gly23 by
Asn, Ile 24 by Asn, Phe 36 by Asn, Asp 37 by Asn, or Ser39 by Asn,
or a combination of two or more of these substitutions.
[0018] On the other hand, it may be desirable to provide the
trefoil domain from position 58 to 95 with a glycosylation site
lacking in this domain in native HSP and PSP. Thus, Glu61 may be
replaced by Asn, Asp64 by Asn, Arg66 by Thr or Ser, Gly69 by Thr or
Ser, or Gly72 is replaced by Asn, or a combination of two or more
of these substitutions. In a currently preferred embodiment of the
variant, Asp64 is replaced by Asn, and Arg66 is replaced by Thr or
Ser.
[0019] It is of course understood that variants of the invention
may be glycosylated in the same manner at one or more of these
sites as described above for glycosylation at Asn15 in full-length
HSP.
A DNA sequence encoding HSP may suitably be isolated from a human
genomic DNA library by PCR (polymerase chain reaction) cloning
using primers based on the published cDNA sequence [8].
Alternatively, the DNA sequence may be prepared synthetically by
established standard methods, e.g. the phosphoamidite method
described by S. L. Beaucage and M. H. Caruthers, Tetrahedron
Letters 22, 1981, pp. 1859-1869, or the method described by Matthes
et al., EMBO Journal 3, 1984, pp. 801-805. According to the
phosphoamidite method, oligonucleotides are synthesized, e.g. in an
automatic DNA synthesizer, purified, annealed, ligated and cloned
in suitable vectors. The cDNA sequence shown in [8] may be used as
the basis of oligonucleotide synthesis. Alternatively, it is
possible to use cDNA coding for HSP obtained by screening a human
cDNA library with oligonucleotide probes in accordance with
well-known procedures.
[0020] Furthermore, the DNA sequence may be of mixed synthetic and
genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin
prepared by ligating fragments of genomic, synthetic or cDNA origin
(as appropriate), the fragments corresponding to various parts of
the entire DNA sequence, in accordance with standard
techniques.
[0021] The SP variant of the invention may be encoded by a fragment
of the full-length DNA sequence, prepared by one of the methods
indicated above, or by suitably truncating the full-length
sequence.
[0022] The DNA sequence encoding HSP or an SP variant of the
invention may then be inserted in a suitable expression vector. The
recombinant expression vector may be any vector which may
conveniently be subjected to recombinant DNA procedures, and the
choice of vector will often depend on the host cell into which it
is to be introduced. Thus, the vector may be an autonomously
replicating vector, i.e. a vector which exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g. a plasmid. Alternatively, the vector
may be one which, when introduced into a host cell, is integrated
into the host cell genome and replicated together with the
chromosome(s) into which it has been integrated.
[0023] In the vector, the DNA sequence encoding HSP or an SP
variant of the invention should be operably connected to a suitable
promoter sequence. The promoter may be any DNA sequence which shows
transcriptional activity in the host cell of choice and may be
derived from genes encoding proteins either homologous or
heterologous to the host cell. Examples of suitable promoters for
directing the transcription of the DNA encoding the inhibitor of
the invention in mammalian cells are the SV 40 promoter (Subramani
et al., Mol. Cell. Biol. 1, 1981, pp. 854-864), the MT-1
(metallothionein gene) promoter (Palmiter et al., Science 222,
1983, pp. 809-814) or the adenovirus 2 major late promoter.
Suitable promoters for use in yeast host cells include promoters
from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255,
1980, pp. 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1,
1982, pp. 419-434) or alcohol dehydrogenase genes (Young et al., in
Genetic Engineering of Microorganisms for Chemicals (Hollaender et
al, eds.), Plenum Press, New York, 1982), or the TPI1 (U.S. Pat.
No. 4,599,311) or ADH2-4-c (Russell et al., Nature 304, 1983, pp.
652-654) promoters. Suitable promoters for use in filamentous
fungus host cells are, for instance, the ADH3 promoter (McKnight et
al., The EMBO J. 4, 1985, pp. 2093-2099) or the tpiA promoter.
[0024] The DNA sequence encoding HSP or an SP variant may also be
operably connected to a suitable terminator, such as the human
growth hormone terminator (Palmiter et al., op. cit.) or (for
fungal hosts) the TPI1 (Alber and Kawasaki, op. cit.) or ADH3
(McKnight et al., op. cit.) promoters. The vector may further
comprise elements such as polyadenylation signals (e.g. from SV 40
or the adenovirus 5 Elb region), transcriptional enhancer sequences
(e.g. the SV 40 enhancer) and translational enhancer sequences
(e.g. the ones encoding adenovirus VA RNAs).
[0025] The recombinant expression vector may further comprise a DNA
sequence enabling the vector to replicate in the host cell in
question. An examples of such a sequence (when the host cell is a
mammalian cell) is the SV 40 origin of replication, or (when the
host cell is a yeast cell) the yeast plasmid 2.mu. replication
genes REP 1-3 and origin of replication. The vector may also
comprise a selectable marker, e.g. a gene the product of which
complements a defect in the host cell, such as the gene coding for
dihydrofolate reductase (DHFR) or one which confers resistance to a
drug, e.g. neomycin, hygromycin or methotrexate, or the
Schizosaccharomyces pombe TPI gene (described by P. R. Russell,
Gene 40, 1985, pp. 125-130.
[0026] The procedures used to ligate the DNA sequences coding for
HSP or the SP variant, the promoter and the terminator,
respectively, and to insert them into suitable vectors containing
the information necessary for replication, are well known to
persons skilled in the art (cf., for instance, Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,
1989).
[0027] The host cell into which the expression vector is introduced
may be any cell which is capable of producing the inhibitor of the
invention and is preferably a eukaryotic cell, such as a mammalian,
yeast or fungal cell.
[0028] The yeast organism used as the host cell may be any yeast
organism which, on cultivation, produces large quantities of the
inhibitor of the invention. Examples of suitable yeast organisms
are strains of the yeast species Saccharomyces cerevisiae,
Saccharomyces kluyveri, Schizosaccharomyces pombe or Saccharomyces
uvarum. The transformation of yeast cells may for instance be
effected by protoplast formation followed by transformation in a
manner known per se.
[0029] Examples of suitable mammalian cell lines are the COS (ATCC
CRL 1650), BHK (ATCC CRL 1632, ATCC CCL 10) or CHO (ATCC CCL 61)
cell lines. Methods of transfecting mammalian cells and expressing
DNA sequences introduced in the cells are described in e.g. Kaufman
and Sharp, J. Mol. Biol. 159, 1982, pp. 601-621; Southern and Berg,
J. Mol. Appl. Genet. 1, 1982, pp. 327-341; Loyter et al., Proc.
Natl. Acad. Sci. USA 79, 1982, pp. 422-426; Wigler et al., Cell 14,
1978, p. 725; Corsaro and Pearson, Somatic Cell Genetics 7, 1981,
p. 603, Graham and van der Eb, Virology 52, 1973, p. 456; and
Neumann et al., EMBO J. 1, 1982, pp. 841-845.
[0030] Alternatively, fungal cells may be used as host cells.
Examples of suitable fungal cells are cells of filamentous fungi,
e.g. Aspergillus spp. or Neurospora spp., in particular strains of
Aspergillus oryzae or Aspergillus niger. The use of Aspergillus
spp. for the expression of proteins is described in, e.g., EP 238
023.
[0031] According to the present method, yeast cells are currently
preferred for producing HSP and other SPs (such as those shown in
FIG. 1), as they have surprisingly been found to produce SP in a
high yield and in at least 60% glycosylated form. For instance,
about two thirds of the HSP produced by yeast may be recovered in
glycosylated form.
[0032] The medium used to cultivate the cells may be any
conventional medium suitable for growing mammalian cells or fungal
(including yeast) cells, depending on the choice of host cell. The
spasmolytic polypeptide will be secreted by the host cells to the
growth medium and may be recovered therefrom by conventional
procedures including separating the cells from the medium by
centrifugation or filtration, precipitating the proteinaceous
components of the supernatant or filtrate by means of a salt, e.g.
ammonium sulfate, purification by a variety of chromatographic
procedures, e.g. ion exchange chromatography or affinity
chromatography, or the like.
[0033] The present invention also relates to a pharmaceutical
composition comprising HSP or a variant spasmolytic polypeptide of
the invention together with a pharmaceutically acceptable carrier
or excipient. In the composition of the invention, the variant may
be formulated by any of the established methods of formulating
pharmaceutical compositions, e.g. as described in Remington's
Pharmaceutical Sciences, 1985. The composition may typically be in
a form suited for oral or rectal administration and may, as such,
be formulated as tablets or suppositories.
[0034] HSP or an SP variant of the invention is contemplated to be
useful for the prophylaxis or treatment of gastrointestinal
disorders. More specifically, it is contemplated for the treatment
of gastric or peptic ulcers, inflammatory bowel disease, Crohn's
disease or injury to the intestinal tract caused by radiation
therapy, bacterial or other infections, etc.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The invention is further illustrated in the following
example with reference to the appended drawings in which
[0036] FIG. 1 shows the trefoil family of peptides. Intestinal
trefoil factor (ITF) contains one trefoil domain [2], as does the
breast cancer associated pS2 peptide [3, 4]. The spasmolytic
polypeptides from man, pig and mouse contain two trefoil domains
[1, 8]. Spasmolysins from Xenopus laevis contain one or four
trefoil domains [10]. Recently, a member of the frog trefoil family
containing two domains has been described [11].
[0037] FIG. 2 shows the proposed structure of human spasmolytic
polypeptide, HSP. The primary amino acid sequence is taken from
Tomasetto et al. [8], and the disulphide bonds are placed in
homology to PSP [1].
[0038] FIG. 3 shows the nucleotide sequence (SEQ ID NO:2) and
corresponding amino acid sequence (SEQ ID NO: 3) of the 563 bp
EcoRI-XbaI fragment encoding the leader-HSP fusion protein. The Kex
2 processing site is indicated by a vertical arrow. The leader and
the PCR cloned parts of the HSP gene are shown in capital letters,
while the synthetic parts are shown in small letters. The
underlined sequences correspond to the PCR primers with horizontal
arrows indicating the direction. Restriction sites relevant for the
construction are shown.
[0039] FIG. 4 shows the S. cerevisiae plasmid for the expression
and secretion of HSP. TPI-prom. and TPI-term. are S. cerevisiae
triosephosphate isomerase transcription promoter and terminator
sequences, respectively. POT is a selective marker, the
Schizosaccharomyces pombe triosephosphate isomerase gene. Only
restriction sites relevant for the construction of the plasmid have
been indicated.
[0040] FIG. 5 shows reversed-phase HPLC on a Vydac 214TP54 column
of yeast fermentation broth. The two peaks corresponding to r-HSP
and glycosylated r-HSP are indicated. The dashed line shows the
concentration of acetonitrile in the eluting solvent.
[0041] FIG. 6 shows ion exchance chromatography on a Fast Flow S
column of concentrated yeast supernatant. The amount of r-HSP and
glycosylated r-HSP were determined by the use of the HPLC system
shown in FIG. 5. The bars indicate the fractions pooled for further
purification of r-HSP and glycosylated r-HSP. The dashed line shows
the concentration of NaCl in the eluting solvent. For details, see
Material and Methods.
[0042] FIG. 7 shows the final purification of r-HSP (A) and
glycosylated r-HSP (B) on a preparative reversed-phase HPLC Vydac
214TP1022 column. The bars indicate the fractions pooled for
lyophilization. The dashed lines show the concentration of
acetonitrile in the eluting solvent. For details, see Material and
Methods.
[0043] FIG. 8 shows reversed-phase HPLC on a Vydac 214TP54 column
of purified, glycosylated r-HSP (A) and r-HSP (B). The dashed lines
show the concentration of acetonitrile in the eluting solvent.
[0044] FIG. 9 shows mass spectra of purified r-HSP (A and B) and
glycosylated r-HSP (C and D). Fig. A and Fig. C show the original
mass spectrum of r-HSP and glycosylated r-HSP, respectively. Fig. B
and Fig. D show the reconstructed mass spectrum for r-HSP and
glycosylated r-HSP on the basis of Fig. A and Fig. C.
EXAMPLE
Material and Methods
General Methods
[0045] Standard DNA techniques were used as previously described
[29]. Synthetic oligonucleotides were prepared on an automatic DNA
synthesizer (380B, Applied Biosystems) using commercially available
reagents. DNA sequence determinations were performed by the dideoxy
chain-termination technique [30]. Polymerase chain reactions (PCR)
were performed on a DNA Thermal Cycler (Perkin Elmer Cetus) using a
commercial kit (GeneAmp, Perkin Elmer Cetus).
PCR Cloning of HSP
[0046] The first trefoil domain of HSP was isolated by a PCR
reaction in which 1 .mu.g human genomic DNA (Clontech, Palo Alto,
Calif., USA) was used as a template. The reaction mixture contained
100 pmole each of the forward primer 1 (GGCTGAGCCCCCATAACAG) (SEQ
ID NO:4) and reverse primer 2 (TGGAAACACCAGGGGAC) (SEQ ID NO:5)
(FIG. 3) and was carried out in a 100 .mu.l volume. The cycle was:
94.degree. C. for 1 min, 50.degree. C. for 1 min, and 72.degree. C.
for 1 min. After 30 cycles a final cycle was performed in which the
72.degree. C. step was maintained for 10 min. The PCR product, a
115 bp fragment, was isolated by electrophoresis on a 2% agarose
gel.
[0047] The 115 bp PCR fragment was digested with DdeI and then
ligated to a 31 bp duplex formed from the oligonucleotides
(GAGAAACCCTCCCCCTGCCAGTGCTCCAGGC) (SEQ ID NO:6) and
(TCAGCCTGGAGCACTGGCAGGGGGAGGGTTTCTC). The ligation product was
amplified by PCR using forward primer 3
(GCTGAGAGATTGGAGAAGAGAGAGAAACCCTCCCCCT) (SEQ ID NO:7) and reverse
primer 2. The 3' part of primer 3 is identical to the N-terminal
encoding part of the HSP gene and the 5' part of primer 3 is
identical to the C-terminal encoding part of the hybrid leader gene
(FIG. 3). In-frame fusion of the hybrid leader gene and the first
trefoil domain from HSP was obtained by overlay extension PCR [31].
The product was digested with EcoRI and AvaII and isolated as a 360
bp DNA fragment.
[0048] The second trefoil domain of HSP was PCR-cloned from human
genomic DNA as described for the first domain by replacing primers
1 and 2 with forward primer 4 (TGCGTCATGGAGGTCTC) (SEQ ID NO:8) and
reverse primer 5 (AGCACCATGGCACTTCAAAG) (SEQ ID NO:9) (FIG. 3).
Reverse primer 5 introduces a NcoI site as a silent mutation. The
PCR product, a 115 bp fragment, was isolated and digested with DdeI
and NcoI resulting in a 91 bp fragment. To this fragment were
ligated two synthetic duplexes. The first, encoding the amino acid
sequence between the two trefoil domains, consisted of the
oligonucleotides
(GTCCCCTGGTGTTTCCACCCCCTCCCAAAGCAAGAGTCGGATCAGTGCGTCATGGAGGTC) (SEQ
ID NO:10) and
(TGAGACCTCCATGACGCACTGATCCGACTCTTGCTTTGGGAGGGGGTGGAAACACCAGGG) (SEQ
ID NO:11). The second, a 46 bp NcoI-XbaI fragment encoding the
C-terminal part of HSP, consisted of the oligonucleotides
(CATGGTGCTTCTTCCCGAACTCTGTGGAAGACTGCCATTACTAAGT) (SEQ ID NO:12) and
(CTAGACTTAGTAATGGCAGTCTTCCACAGAGTTCGGGAAGAAGCAC) (SEQ ID NO:13).
After AvaII digestion a 195 bp AvaII-XbaI fragment was
isolated.
[0049] A DNA construct encoding the hybrid leader fused in-frame to
the entire HSP gene was obtained by ligation of the 360 bp
EcoRI-AvaII fragment and the 195 bp AvaII-XbaI fragment described
above to the 2.7 kb EcoRI-XbaI fragment from vector pTZ19R [32].
This construct was then transformed into E. coli strain MT-172
(r.sup.-, m.sup.+) by selection for resistance to ampicillin. DNA
sequencing of the resulting plasmid, KFN-1843, showed that the
correct construction had been obtained.
Construction of the HSP Secreting Yeast Strain
[0050] Plasmid KFN-1843 described above was digested with EcoRI and
XbaI. The resulting 558 bp fragment was isolated and ligated to the
9.3 kb NcoI-XbaI fragment and the 1.6 kb NcoI-EcoRI fragment both
from the yeast expression vector pMT-636. Plasmid pMT-636 is
derived from the S. cerevisiae-E. coli shuttle vector CPOT [25, 33]
by deletion of the 0.4 kb HpaI-NruI fragment from the Leu-2 gene.
The ligation mixture was trans-formed into E. coli strain MT-172,
and the HSP expression plasmid, KFN-1847, was isolated (FIG. 4).
Plasmid pKFN-1847 was transformed into S. cerevisiae strain MT-663
by selection for growth on glucose as the sole carbon source. One
transformant, KFN-1852, was selected for fermentation.
Fermentation
[0051] The transformant described above was cultivated at
30.degree. C. for 3 days in yeast peptone dextrose (YPD) medium
[40] supplied with additional yeast extract (60 g/l). An OD 650 nm
value of 52 was reached at the end of the fermentation.
Purification of r-HSP
[0052] The concentration of r-HSP in the yeast fermentation broth
and fractions obtained during the purification was measured by
analytical HPLC. Aliquots (usually 50-200 .mu.l) were injected onto
a Vydac 214TP54 reverse-phase C4 HPLC column (0.46.times.25 cm)
equilibrated at 30.degree. C. at a flow rate of 1.5 ml/min with
0.1% (v/v) TFA in 5% (v/v) acetonitrile. The concentration of
acetonitrile in the eluting solvent was raised to 65% (v/v) over 30
min. Absorbance was measured at 280 nm. The peaks eluting at 15.6
min. and 16.1 min. (FIG. 5) was found by mass spectrometry analysis
to represent glycosylated r-HSP and unglycosylated r-HSP,
respectively. The peptides were quantified using a calibrated PSP
sample as standard as both peptides contain two Trp and two Tyr out
of 106 amino acid residues.
[0053] From a 10 litre fermentor, 8 litres of fermentation broth
was isolated by centrifugation at 3,000 rpm for 10 min. The
supernatant was concentrated to 0.9 litre using an Amicon
ultrafiltration unit (RA 2000) equipped with an Amicon spiral
ultrafiltration cartridge type S1Y3, MW cutoff 3,000 (Product No.
540620). The pH was adjusted to 1.7 and the conductivity in the
resulting concentrated sample was measured to 4.7 mS.
[0054] The sample was pumped onto a Fast Flow S-Sepharose
(Pharmacia) column (5.times.11 cm) with a flow rate of 40 ml/h.
Previous to the application, the column was equilibrated in 50 mM
formic acid buffer, pH=3.7. After application of the sample, the
column was washed with 500 ml of 50 mM formic acid buffer, pH=3.7.
The peptides were eluted from the column by a linear gradient
between 1.5 litres of 50 mM formic acid buffer, pH=3.7 and 1.5
litres of 50 mM formic acid buffer, pH=3.7 containing 0.6 M NaCl.
Fractions of 10 ml was collected at a flow rate of 40 ml/h and the
absorbance was measured at 280 nm. Fractions were assayed for the
content of r-HSP and glycosylated r-HSP in the HPLC-system
previously described. The elution profile is shown in FIG. 6.
Fractions corresponding to r-HSP (fract. Nos. 107-128) and
glycosylated r-HSP (fract. Nos. 78-95), respectively, were
pooled.
[0055] Glycosylated r-HSP and r-HSP were further purified by
preparative HPLC chromatography. Pooled fractions (approx. 200 ml)
were pumped onto a Vydac 214TP1022 C4 column (2.2.times.25 cm)
equilibrated in 0.1% (v/v) TFA. The column was washed with 100 ml
of 0.1% (v/v) TFA in 10% (v/v) MeCN. The peptides were eluted at
25.degree. C. and at a flow rate of 5 ml/min with a linear gradient
(650 ml) formed from MeCN/H.sub.2O/TFA (10.0:89.9:0.1 v/v/v) and
MeCN/H.sub.2O/TFA (60.0:39.9:0.1 v/v/v). UV-absorption was
monitored at 280 nm, and fractions corresponding to 10 ml were
collected and analysed for the content of r-HSP or glycosylated
r-HSP. FIG. 7 shows the preparative HPLC purification of r-HSP
(FIG. 7A) and glycosylated r-HSP (FIG. 7B). Fractions corresponding
to the bars were pooled, and the volume reduced to 30% by vacuum
centrifugation. From the two resulting pools, r-HSP and
glycosylated r-HSP were isolated by lyophilization.
Characterization of r-HSP and Glycosylated r-HSP
[0056] Amino acid composition analysis were carried out by
hydrolysis of 50 .mu.g peptide with 6M HCl for 24 h at 110.degree.
C. as previously described [6]; no correction for loss during
hydrolysis was carried out. Amino acid sequence analysis was
determined by automated Edman degradation using an Applied
Biosystems Model 470A gas-phase sequencer [22]. Carbohydrate
composition analysis was carried out by hydrolysis of 50 .mu.g
peptide with 2M HCl for 1 h, 2 h and 4 h at 100.degree. C. and
monosaccharides were separated on a CarboPac PAI (Dionex,
Sunnyvale, Calif.) column (4.times.250 mm) eluted with 14 mM NaOH.
The monosaccharides were detected by pulsed amperometric detection
(Dionex PAD-detector). The amount of monosaccharides was corrected
to zero time of hydrolysis and calculated as nmol of monosaccharide
per nmol of peptide.
[0057] Mass spectrometry analysis was performed using an API III
LC/MS/MS system (Sciex, Thornhill, Ontario, Canada). The triple
quadrupole instrument has a mass-to-charge (m/z) range of 2400 and
is fitted with a pneumatically assisted electrospray (also referred
to as ion-spray) interface [23, 24]. Sample introduction was done
by a syringe infusion pump (Sage Instruments, Cambridge, Mass.)
through a fused capillary (75 .mu.m i.d.) with a liquid flow-rate
set at 0.5-1 .mu.l/min. The instrument m/z scale was calibrated
with the singly-charged ammonium adduct ions of poly(propylene
glycols) (PPG's) under unit resolution.
[0058] The accuracy of mass measurements was generally better than
0.02%.
Results
Expression and Purification
[0059] DNA fragments encoding the two trefoil domains of HSP were
isolated by PCR from human genomic DNA using primers based on the
published cDNA sequence [8]. The full length HSP gene was obtained
from the PCR cloned fragments by addition of synthetic DNA
fragments. The HSP gene was fused in-frame to a hybrid yeast leader
sequence by overlay extension PCR [31] (FIG. 3). The hybrid leader
is based on the mouse salivary amylase signal peptide [34] and the
S. kluvveri .alpha. mating factor leader sequence [35] and is
further modified near the Kex 2 cleavage site for efficient
processing [36, 41].
[0060] The yeast expression plasmid pKFN-1847 contains the
leader-HSP gene inserted between the S. cerevisiae triose phosphate
isomerase promoter and terminator [37]. The expression vector (FIG.
4) also contains the Schizosaccharomyces pombe TPI gene (POT)
[38].
[0061] The plasmid was transformed into the yeast strain MT-663,
carrying a deletion in the TPI gene, by selecting for growth on
glucose.
[0062] The expression level of r-HSP in the present yeast system is
approx. 120 mg/l. As can be seen from FIG. 5, the yeast supernatant
contains two forms of r-HSP; one eluting at R.sub.t=15.6 min. and
one eluting at R.sub.t=16.1 min. These two forms were purified
separately, and by using the analytical HPLC-system (FIG. 5), these
two forms can be quantified individually during the different steps
of the purification.
[0063] After the initial concentration of the yeast supernatant by
ultrafiltration, the first purification step was cationic exchange
chromatography on a Fast Flow S column. FIG. 6 shows the elution
profile from the column including the amount of r-HSP and
glycosylated r-HSP determined in the fractions. A complete
separation of the two forms of r-HSP was obtained in this step.
[0064] The fractions from the Fast Flows S column were pooled as
indicated in FIG. 6, and the two peptides were further purified by
preparative HPLC (FIG. 7). The r-HSP and glycosylated r-HSP were
recovered from the fractions indicated in FIG. 7A and FIG. 7B by
vacuum centrifugation and lyophilization. The purification is
summarized in Table 1. The overall yield of r-HSP and glycosylated
r-HSP from 8 litres of fermentation broth was 160 mg and 219 mg
corresponding to 50% and 34%, respectively.
Characterization of r-HSP and Glycosylated r-HSP
[0065] FIG. 8 shows the purity of r-HSP and glycosylated r-HSP as
analysed by analytical HPLC. From these results none of the
peptides looks completely pure. However, upon rechromatography of
material eluting in the minor as well as the major peak, similar
chromatograms were obtained for both peptides (results not shown).
This seems to indicate that the double peak observed for both r-HSP
and glycosylated r-HSP reflects an atypical behaviour of these
peptides on reverse phase columns rather than impurities in the
preparations.
[0066] Table 2 shows the amino acid sequencing results obtained on
r-HSP and glycosylated r-HSP. The average repetitive yield was
94.4% (r-HSP) and 94.6% (glycosylated r-HSP), respectively. In both
cases the first 40 residues of the two peptides were confirmed by
the sequence analysis. In the glycosylated HSP, no PTH-a.a. was
found in Edman degradation cycle No. 15. The HSP sequence from
residue 15-17 (Asn-Arg-Thr) corresponds to a consensus sequence for
N-glycosylation of Asn-15.
[0067] The carbohydrate composition analysis of glysocylated r-HSP
showed the presence of 12.8 nmol mannose (Man) and 1.6 nmol of
N-acetyl glucoseamine (GlcNAc) per nmol of r-HSP. By peptide
mapping of r-HSP and glycosylated r-HSP in combination with mass
spectrometry and sequencing analysis (results not shown), no other
residue besides Asn-15 of the glycosylated r-HSP was found to be
modified, i.e. no O-glycosylation was found.
[0068] In FIG. 9, the electro-spray mass spectrometry (ESMS)
analysis is shown for r-HSP and glycosylated r-HSP. FIGS. 9A and 9C
are original mass spectra displaying characteristics series of
multiply charged protonated ions always observed in ESMS spectra of
proteins. FIGS. 9B and 9D are the corresponding computer
reconstructed mass spectra from which the molecular weight of
individual components may be read directly. As can be seen from
FIG. 9B, the MW found for r-HSP is 11961.5.+-.2 which is in very
good agreement with a calculated mass of 11961.3. FIG. 9D shows the
reconstructed ion spray mass spectrum of the glycosylated r-HSP.
From the sequence analysis and the carbohydrate composition
analysis, it is known that only Asn-15 is glycosylated and that
only two monosaccharide residues, mannose and N-acetyl
glucoseamine, occur in the glycosylated form of r-HSP. From these
results in combination with the mass spectrometry data, it is
possible to deduce the different glycosylated forms of r-HSP (Table
3).
[0069] Molecular weights corresponding to two series of
carbohydrate side chains can be deduced from the combination of
carbohydrate composition data and ISMS-data, namely
(GlcNAc).sub.2(Hex).sub.10-15 and (Hex).sub.13-17 (Table 3). As
mannose is the only hexose in the glycosylated r-HSP, and as Asn-15
is the only glycosylated residue, it seems reasonable to conclude
that the structure of the glycosylation site is
Asn-(GlcNAc).sub.2-(Man).sub.10-15. The observed
Asn-(Hex).sub.13-17 forms are thus most likely to arise from
fragmentation in the mass spectrometer, by which the two GlcNAc
residues lose an acetyl group and are converted into two
hexoses.
[0070] The structure of Asn-(GlcNAc).sub.2-(Man).sub.10-15 has
previously been reported as high mannose type of N-glycosylation
for other peptides and proteins expressed in yeast [26].
TABLE-US-00002 TABLE 1 Purification of r-hSP and glycosylated r-hSP
from yeast supernatant Vol- Amount [mg] Yield [%] ume r-
glycosylated r- glycosylated STEP [ml] hSP r-hSP hSP r-hSP Yeast
8000 320 640 100 100 supernatant Ultrafiltration 900 207 405 65 63
Ion exchange Pool 1 160 275 43 chroma- Pool 2 220 182 57 tography
Prep HPLC Pool 1 54 219 34 Pool 2 80 160 50
TABLE-US-00003 TABLE 2 Amino acid sequence analysis of r-hSP and
glycosylated r-hSP Yield (pmol) glycosylated r- Cycle No. PTH-a.a.
r-hSP hSP 1 Glu 4304 8853 2 Lys 6925 8292 3 Pro 6027 12837 4 Ser
2890 5602 5 Pro 4336 8802 6 (Cys) ND ND 7 Gln 3388 5689 8 (Cys) ND
ND 9 Ser 1279 2417 10 Arg 1876 2523 11 Leu 2277 4290 12 Ser 877
1790 13 Pro 1545 2963 14 His 517 574 15 Asn 1202 0* 16 Arg 959 1471
17 Thr 978 2172 18 Asn 1066 1509 19 (Cys) ND ND 20 Gly 836 1857 21
Phe 993 1958 22 Pro 843 1839 23 Gly 785 2049 24 Ile 640 1400 25 Thr
589 1454 26 Ser 274 621 27 Asp 581 1391 28 Gln 445 952 29 (Cys) ND
ND 30 Phe 623 1562 31 Asp 483 1210 32 Asn 369 823 33 Gly 359 885 34
(Cys) ND ND 35 (Cys) ND ND 36 Phe 422 1094 37 Asp 268 783 38 Ser
127 324 39 Ser 145 394 40 Val 298 827 ND: Not determined *No trace
of PTH-Asn or PTH-Asp was seen in cycle No. 15 of glycosylated
r-hSP.
TABLE-US-00004 TABLE 3 Mass analysis of glycosylated r-hSP
Calculated MW found by Structure MW ESMS (FIG. 9D) hSP + 2 GlcNAc +
10 Man 13989.1 13989.5 hSP + 2 GlcNAc + 11 Man 14151.2 14151.0 hSP
+ 2 GlcNAc + 12 Man 14313.4 14313.5 hSP + 2 GlcNAc + 13 Man 14475.5
14475.0 hSP + 2 GlcNAc + 14 Man 14639.7 14639.5 hSP + 2 GlcNAc + 15
Man 14799.8 14801.5 hSP + 13 Man 14069.1 14072.0 hSP + 14 Man
14231.3 14232.5 hSP + 15 Man 14393.4 14393.0 hSP + 16 Man 14555.5
14557.5 hSP + 17 Man 14717.7 14720.0
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Sequence CWU 1
1
141106PRTHomo Sapien 1Glu Lys Pro Ser Pro Cys Gln Cys Ser Arg Leu
Ser Pro His Asn Arg 1 5 10 15Thr Asn Cys Gly Phe Pro Gly Ile Thr
Ser Asp Gln Cys Phe Asp Asn 20 25 30Gly Cys Cys Phe Asp Ser Ser Val
Thr Gly Val Pro Trp Cys Phe His 35 40 45Pro Leu Pro Lys Gln Glu Ser
Asp Gln Cys Val Met Glu Val Ser Asp 50 55 60Arg Arg Asn Cys Gly Tyr
Pro Gly Ile Ser Pro Glu Glu Cys Ala Ser65 70 75 80Arg Lys Cys Cys
Phe Ser Asn Phe Ile Phe Glu Val Pro Trp Cys Phe 85 90 95Phe Pro Asn
Ser Val Glu Asp Cys His Tyr 100 1052563DNAHomo
SapienCDS(77)...(553)sig_peptide(77)...(235)mat_peptide(236)...(553)
2gaattccatt caagaatagt tcaaacaaga agattacaaa ctatcaattt catacacaat
60ataaacgacc aaaaga atg aag gct gtt ttc ttg gtt ttg tcc ttg atc gga
112 Met Lys Ala Val Phe Leu Val Leu Ser Leu Ile Gly -50 -45ttc tgc
tgg gcc caa cca gtc act ggc gat gaa tca tct gtt gag att 160Phe Cys
Trp Ala Gln Pro Val Thr Gly Asp Glu Ser Ser Val Glu Ile -40 -35
-30ccg gaa gag tct ctg atc atc gct gaa aac acc act ttg gct aac gtc
208Pro Glu Glu Ser Leu Ile Ile Ala Glu Asn Thr Thr Leu Ala Asn
Val-25 -20 -15 -10gcc atg gct gag aga ttg gag aag aga gag aaa ccc
tcc ccc tgc cag 256Ala Met Ala Glu Arg Leu Glu Lys Arg Glu Lys Pro
Ser Pro Cys Gln -5 1 5tgc tcc agg ctg agc ccc cat aac agg acg aac
tgc ggc ttc cct gga 304Cys Ser Arg Leu Ser Pro His Asn Arg Thr Asn
Cys Gly Phe Pro Gly 10 15 20atc acc agt gac cag tgt ttt gac aat gga
tgc tgt ttc gac tcc agt 352Ile Thr Ser Asp Gln Cys Phe Asp Asn Gly
Cys Cys Phe Asp Ser Ser 25 30 35gtc act ggg gtc ccc tgg tgt ttc cac
ccc ctc cca aag caa gag tcg 400Val Thr Gly Val Pro Trp Cys Phe His
Pro Leu Pro Lys Gln Glu Ser 40 45 50 55gat cag tgc gtc atg gag gtc
tca gac aga aga aac tgt ggc tac ccg 448Asp Gln Cys Val Met Glu Val
Ser Asp Arg Arg Asn Cys Gly Tyr Pro 60 65 70ggc atc agc ccc gag gaa
tgc gcc tct cgg aag tgc tgc ttc tcc aac 496Gly Ile Ser Pro Glu Glu
Cys Ala Ser Arg Lys Cys Cys Phe Ser Asn 75 80 85ttc atc ttt gaa gtg
cca tgg tgc ttc ttc ccg aac tct gtg gaa gac 544Phe Ile Phe Glu Val
Pro Trp Cys Phe Phe Pro Asn Ser Val Glu Asp 90 95 100tgc cat tac
taagtctaga 563Cys His Tyr 1053159PRTHomo SapienSIGNAL(1)...(53)
3Met Lys Ala Val Phe Leu Val Leu Ser Leu Ile Gly Phe Cys Trp Ala
-50 -45 -40Gln Pro Val Thr Gly Asp Glu Ser Ser Val Glu Ile Pro Glu
Glu Ser -35 -30 -25Leu Ile Ile Ala Glu Asn Thr Thr Leu Ala Asn Val
Ala Met Ala Glu -20 -15 -10Arg Leu Glu Lys Arg Glu Lys Pro Ser Pro
Cys Gln Cys Ser Arg Leu-5 1 5 10Ser Pro His Asn Arg Thr Asn Cys Gly
Phe Pro Gly Ile Thr Ser Asp 15 20 25Gln Cys Phe Asp Asn Gly Cys Cys
Phe Asp Ser Ser Val Thr Gly Val 30 35 40Pro Trp Cys Phe His Pro Leu
Pro Lys Gln Glu Ser Asp Gln Cys Val 45 50 55Met Glu Val Ser Asp Arg
Arg Asn Cys Gly Tyr Pro Gly Ile Ser Pro60 65 70 75Glu Glu Cys Ala
Ser Arg Lys Cys Cys Phe Ser Asn Phe Ile Phe Glu 80 85 90Val Pro Trp
Cys Phe Phe Pro Asn Ser Val Glu Asp Cys His Tyr 95 100
105419DNASynthetic 4ggctgagccc ccataacag 19517DNASynthetic
5tggaaacacc aggggac 17631DNASynthetic 6gagaaaccct ccccctgcca
gtgctccagg c 31734DNASynthetic 7tcagcctgga gcactggcag ggggagggtt
tctc 34837DNASynthetic 8gctgagagat tggagaagag agagaaaccc tccccct
37917DNASynthetic 9tgcgtcatgg aggtctc 171020DNASynthetic
10agcaccatgg cacttcaaag 201160DNASynthetic 11gtcccctggt gtttccaccc
cctcccaaag caagagtcgg atcagtgcgt catggaggtc 601260DNASynthetic
12tgagacctcc atgacgcact gatccgactc ttgctttggg agggggtgga aacaccaggg
601346DNASynthetic 13catggtgctt cttcccgaac tctgtggaag actgccatta
ctaagt 461446DNASynthetic 14ctagacttag taatggcagt cttccacaga
gttcgggaag aagcac 46
* * * * *