Fusion protein and application thereof
阅读说明：本技术 融合蛋白及其应用 (Fusion protein and application thereof ) 是由 龚珉 于 2020-04-15 设计创作，主要内容包括：本申请提供了一种融合蛋白,其包含活性蛋白、连接子和多肽；其中,所述多肽包含S～(1)和S～(2),其中沿N端至C端方向上S～(1)和S～(2)以肽键串联连接；所述S～(1)为如SEQ ID No:1所示的氨基酸序列或其功能性变体；所述S～(2)为如SEQ ID No:7所示的氨基酸序列或其功能性变体。(The present application provides a fusion protein comprising an active protein, a linker, and a polypeptide; wherein the polypeptide comprises S 1 And S 2 Wherein S is along the direction from N terminal to C terminal 1 And S 2 Are connected in series by peptide bonds; said S 1 Is an amino acid sequence shown as SEQ ID No. 1 or a functional variant thereof; said S 2 Is an amino acid sequence shown as SEQ ID No. 7Or a functional variant thereof.)
1. A fusion protein comprising an active protein, a linker, and a polypeptide; wherein the content of the first and second substances,
the polypeptide comprises S1And S2Wherein
S along the direction from N terminal to C terminal1And S2Are connected in series by peptide bonds;
said S1Is an amino acid sequence shown as SEQ ID No. 1 or a functional variant thereof;
said S2Is an amino acid sequence as shown in SEQ ID No. 7 or a functional variant thereof;
preferably, the polypeptide is an amino acid sequence shown as SEQ ID No. 2.
2. The fusion protein of claim 1, wherein the polypeptide comprises S1、S2And S3Wherein
S along the direction from N terminal to C terminal1、S2And S3Are connected in series by peptide bonds;
said S1Is an amino acid sequence shown as SEQ ID No. 1 or a functional variant thereof;
said S2Is an amino acid sequence as shown in SEQ ID No. 7 or a functional variant thereof;
said S3Is an amino acid sequence shown as SEQ ID No. 8 or a functional variant thereof;
preferably, the polypeptide is an amino acid sequence shown as SEQ ID No. 3 or SEQ ID No. 4.
3. The fusion protein of claim 1, wherein the polypeptide comprises S1、X1And S2Wherein
S along the direction from N terminal to C terminal1、S2、X1Are connected in series with each other by peptide bondsConnecting;
said S1Is an amino acid sequence shown as SEQ ID No. 1 or a functional variant thereof;
said S2Is an amino acid sequence as shown in SEQ ID No. 7 or a functional variant thereof;
said X1Is any amino acid, preferably lysine or arginine, more preferably arginine;
optionally, the polypeptide is other than X1And S2Comprising in addition two S1Preferably, the polypeptide is an amino acid sequence shown as SEQ ID No. 5 or SEQ ID No. 6.
4. The fusion protein of claim 1, wherein
The active protein, the linker and the polypeptide are sequentially arranged along the direction from the N end to the C end; or the active protein, the linker and the polypeptide are sequentially arranged in the sequence from the N end to the C end; and/or
The active protein is a protein drug; preferably, the active protein is selected from one or more of the following: GLP-1, EPO, Eryysin A, Exendin-4, PKA competitive polypeptide inhibitors, insulin, monoclonal antibody drugs or fragments thereof; and/or
The linker comprises a repeat sequence such as GGGS or TP; preferably, the linker is an amino acid sequence as shown in any one of SEQ ID Nos. 13-16 or a functional variant thereof.
5. The fusion protein of claim 1, wherein the fusion protein is an amino acid sequence as set forth in any one of SEQ ID nos 9-12.
6. A pharmaceutical composition comprising the fusion protein of any one of claims 1-5 and one or more pharmaceutically acceptable excipients;
preferably, the pharmaceutically acceptable excipients are one or more selected from the group consisting of: soluble filler, pH regulator, stabilizer, non-ionic surfactant and osmotic pressure regulator.
7. Use of the fusion protein of any one of claims 1-5 in the manufacture of a medicament for treating a disease;
preferably, the disease is diabetes, obesity, anemia or non-alcoholic fatty liver disease.
8. A nucleic acid encoding the fusion protein of any one of claims 1-5.
9. An expression system, wherein the expression system is an expression vector or cassette comprising the nucleic acid of claim 8.
10. A cell comprising the fusion protein of any one of claims 1-5 and/or the nucleic acid of claim 8 and/or an expression vector or expression cassette comprising the nucleic acid of claim 8.
Technical Field
The present application relates to the field of pharmaceutical preparation, more specifically, the present application relates to fusion proteins and pharmaceutical compositions comprising the same and uses thereof.
Background
Biological drugs (especially nucleic acid or protein drugs) are widely used for treating various diseases, such as cancer, autoimmune diseases, AIDS and the like. However, biological drugs also have some outstanding problems, such as imperfect drug delivery systems, poor drug stability, short in vivo half-life and low bioavailability. Those skilled in the art will sometimes modify the pharmacokinetic properties of biological drugs to achieve half-life extension, but not all drugs may be modified and some may lose some or all activity during the modification. Therefore, there is an urgent need in the art to find new drug fusion proteins.
Summary of The Invention
In a first aspect, the present application provides a fusion protein comprising an active protein, a linker and a polypeptide, wherein the polypeptide comprises S1And S2Wherein
S along the direction from N terminal to C terminal1And S2Are connected in series by peptide bonds;
said S1Is an amino acid sequence shown as SEQ ID No. 1 or a functional variant thereof;
said S2Is the amino acid sequence shown as SEQ ID No. 7 or functional variant thereof.
In some embodiments, the polypeptide is the amino acid sequence shown in SEQ ID No. 2.
In some embodiments, the polypeptide comprises S1、S2And S3Wherein
S along the direction from N terminal to C terminal1、S2And S3Are connected in series by peptide bonds;
said S1Is an amino acid sequence shown as SEQ ID No. 1 or a functional variant thereof;
said S2Is an amino acid sequence as shown in SEQ ID No. 7 or a functional variant thereof;
said S3Is the amino acid sequence shown as SEQ ID No. 8 or functional variant thereof.
In some embodiments, the polypeptide is the amino acid sequence shown in SEQ ID No. 3 or SEQ ID No. 4.
In some embodiments, the polypeptide comprises S1、X1And S2Wherein
S along the direction from N terminal to C terminal1、S2、X1Are connected in series by peptide bonds;
said S1Is an amino acid sequence shown as SEQ ID No. 1 or a functional variant thereof;
said S2Is an amino acid sequence as shown in SEQ ID No. 7 or a functional variant thereof;
said X1Is any amino acid.
In some embodiments, said X is1Is lysine or arginine.
In some embodiments, said X is1Is arginine.
In some embodiments, the polypeptide is other than X1And S2Comprising in addition two S1。
In some embodiments, the polypeptide is the amino acid sequence shown in SEQ ID No. 5 or SEQ ID No. 6.
In some embodiments, the polypeptide further comprises a pharmaceutically acceptable salt, ester, ether, amide, or mixture thereof.
In some embodiments, the active protein, linker and polypeptide are in that order, in the direction from N-terminus to C-terminus, the active protein, linker and polypeptide.
In some embodiments, the active protein, linker and polypeptide are in that order the polypeptide, linker and active protein in the direction from N-terminus to C-terminus.
In some embodiments, the active protein is a proteinaceous drug.
In some embodiments, the active protein is selected from one or more of the following: GLP-1, EPO, Eryysin A, Exendin-4, PKA competitive polypeptide inhibitors, insulin, monoclonal antibody drugs or fragments thereof.
In some embodiments, the linker comprises a repeat sequence such as GGGS or TP.
In some embodiments, the linker is an amino acid sequence as set forth in any one of SEQ ID Nos 13-16 or a functional variant thereof.
In some embodiments, the fusion protein is an amino acid sequence as set forth in SEQ ID No. 9.
In some embodiments, the fusion protein is an amino acid sequence as set forth in SEQ ID No. 10.
In some embodiments, the fusion protein is an amino acid sequence as set forth in SEQ ID No. 11.
In some embodiments, the fusion protein is an amino acid sequence as set forth in SEQ ID No. 12.
In a second aspect, the present application provides a pharmaceutical composition comprising the fusion protein of the first aspect and one or more pharmaceutically acceptable excipients.
In some embodiments, the pharmaceutically acceptable excipient is one or more selected from the group consisting of: soluble filler, pH regulator, stabilizer, non-ionic surfactant and osmotic pressure regulator.
In a third aspect, the present application provides the use of a fusion protein according to the first aspect in the manufacture of a medicament for the treatment of a disease.
In some embodiments, the disease is diabetes, obesity, anemia, or non-alcoholic fatty liver disease.
In a fourth aspect, the present application provides a nucleic acid encoding the fusion protein of the first aspect.
In a fifth aspect, the present application provides an expression system, wherein:
the expression system is an expression vector or expression cassette,
the expression vector or cassette comprises the nucleic acid of the fourth aspect.
In a sixth aspect, the present application provides a cell comprising the fusion protein of the first aspect and/or the nucleic acid of the fourth aspect and/or an expression vector or cassette comprising the nucleic acid of the fourth aspect.
Drawings
Embodiments of the present application will now be described in detail with reference to the accompanying drawings.
FIG. 1 shows the stability of the fusion protein as shown in SEQ ID No. 9.
FIG. 2 shows the long-acting hypoglycemic function of the fusion protein shown in SEQ ID No: 10.
FIG. 3 shows a solubility comparison of the polypeptides shown in SEQ ID Nos 1-4.
DESCRIPTION OF THE SEQUENCES
SEQ ID No. 1 is S of the present application1An example of an amino acid sequence is the sequence:
QQCTTGQLQCCESTSTANDPATSXLLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV
wherein X is Glu (E) or Lys (K).
SEQ ID No. 2 is a peptide containing S1And S2An example of the polypeptide of (1), which consists of SEQ ID No. 1 and SEQ ID No. 7 at the N-terminus of SEQ ID No. 1. Specifically, the sequence of SEQ ID No. 2 is:
GLTEGLHGFHVHEFGDNTAGSTSAGPRQQCTTGQLQCCESTSTANDPATSXLLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV
wherein X is Glu (E) or Lys (K).
SEQ ID No. 3 is a peptide containing S1、S2And S3An example of the polypeptide of (1), which consists of SEQ ID No. 2 and SEQ ID No. 8 at the N-terminus of SEQ ID No. 2 and X in SEQ ID No. 2 is E. Specifically, the sequence of SEQ ID No. 3 is:
SSMATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGPRQQCTTGQLQCCESTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV
SEQ ID No. 4 is a nucleotide sequence comprising S1、S2And S3An example of the polypeptide of (1), which consists of SEQ ID No. 2 and SEQ ID No. 8 at the N-terminus of SEQ ID No. 2 and X in SEQ ID No. 2 is K. Specifically, the sequence of SEQ ID No. 4 is:
SSMATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGPRQQCTTGQLQCCESTSTANDPATSKLLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV
SEQ ID No. 5 is a peptide containing 2S1、X1And S2An example of the polypeptide of (1), which consists of SEQ ID No:2 and the amino acids R (Arg, arginine) at the C-terminus of SEQ ID No:2 and SEQ ID No:1 and X in SEQ ID No:1 is E and X in SEQ ID No:2 is E. Specifically, the sequence of SEQ ID No. 5 is:
GLTEGLHGFHVHEFGDNTAGSTSAGPRQQCTTGQLQCCESTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNVRQQCTTGQLQCCESTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV
SEQ ID No. 6 is a sequence containing 2S1、X1And S2An example of the polypeptide of (1), which consists of SEQ ID No:2 and the amino acids R (Arg, arginine) at the C-terminus of SEQ ID No:2 and SEQ ID No:1 and X in SEQ ID No:1 is K and X in SEQ ID No:2 is E. Specifically, the sequence of SEQ ID No. 6 is:
GLTEGLHGFHVHEFGDNTAGSTSAGPRQQCTTGQLQCCESTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNVRQQCTTGQLQCCESTSTANDPATSKLLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV
the SEQ ID No. 7 is a polypeptide with the sequence as shown in the specification:
GLTEGLHGFHVHEFGDNTAGSTSAGPR
the SEQ ID No. 8 is a polypeptide with the sequence as shown in the specification:
SSMATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIK
SEQ ID No. 9 is an example of a fusion protein of the present application, wherein the active protein is GLP-1, the linker is an amino acid sequence as shown in SEQ ID No. 13, and the polypeptide is an amino acid sequence as shown in SEQ ID No. 3. Specifically, the sequence of SEQ ID No. 9 is:
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGGGSGGGSGGGSASSMATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGPRQQCTTGQLQCCESTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV
SEQ ID No. 10 is an example of a fusion protein of the present application, wherein the active protein is insulin, the linker is an amino acid sequence as shown in SEQ ID No. 14, and the polypeptide is an amino acid sequence as shown in SEQ ID No. 5. Specifically, the sequence of SEQ ID No. 10 is:
MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCNSGTPTPTPTPTGEFGLTEGLHGFHVHEFGDNTAGSTSAGPRQQCTTGQLQCCESTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNVRQQCTTGQLQCCESTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV
SEQ ID No. 11 is an example of a fusion protein of the present application, wherein the active protein is Eryysin A, the linker is an amino acid sequence as shown in SEQ ID No. 15, and the polypeptide is an amino acid sequence as shown in SEQ ID No. 3. Specifically, the sequence of SEQ ID No. 11 is:
AYAQWVIILIHNVGQQNVKIKNLNASWGKLYADGDKDTEVPASKYEGMVIAPDDQVQINACGREDAAEGTTGTFDLVDPNDSDKQVRHFAWDCPWGTKANSWVVGGSNSKWMIEYTGQNLDSGALGTITVNTLRIGNGGGSGGGSGGGSSSMATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGPRQQCTTGQLQCCESTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV
SEQ ID No. 12 is an example of a fusion protein of the present application, wherein the active protein is EPO, the linker is an amino acid sequence as shown in SEQ ID No. 16, and the polypeptide is an amino acid sequence as shown in SEQ ID No. 3. Specifically, the sequence of SEQ ID No. 12 is:
APPPNLPDPKFESKAALLAARGPEELLCFTERLEDLVCFWEEAASAGVGPGNYSFSYQLEDEPWKLCRLHQAPTARGAVRFWCSLPTADTSSFVPLELRVTAASGAPRYHRVIHINEVVLLDAPVGLVARLADESGHVVLRWLPPPETPMTSHIRYEVDVSAGNGAGSVQRVEILEGRTECVLSNLRGRTRYTFAVRARMAEPSFGGFWSAWSEPVSLLTPSDLDPGGGSGGGSGGGSSSMATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGPRQQCTTGQLQCCESTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV
SEQ ID No. 13 is an example of a linker of the present application, which is a polypeptide having the sequence shown below: GGGSGGGSGGGSA
SEQ ID No. 14 is an example of a linker of the present application, which is a polypeptide having the sequence shown below: SGTPTPTPTPTGEF
SEQ ID No. 15 is an example of a linker of the present application, which is a polypeptide having the sequence shown below: GGGSGGGSGGGS
SEQ ID No. 16 is an example of a linker of the present application, which is a polypeptide having the sequence shown below: PGGGSGGGSGGGS
Detailed Description
While this application contains many specifics, these should not be construed as limitations on the scope of the invention or of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments of particular inventions. Certain features that are described in the context of separate embodiments in this application can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.
Unless otherwise indicated, the terms herein have the same meaning as commonly understood by one of ordinary skill in the art, e.g., in reference to the starting materials and products, operating steps, process parameters, equipment and tools used, and units of values.
Definition of
As used herein, the terms "comprises" and "comprising" mean either open or closed. For example, the term "comprises" or "comprising" may mean that other elements or steps or other elements not listed may also be included or included, or that only the listed elements or steps or other elements may be included or included.
Herein, the term "about" (e.g., in component amounts and reaction parameters) is to be interpreted in a sense that is generally understood by those skilled in the art. In general, the term "about" may be understood as any value within plus or minus 5% of a given value, for example, about X may represent any value in the range of 95% X to 105% X.
In this context, two or more elements are in some way "substantially" identical to one another in the technical requirements and experience of a person skilled in the art in a particular practice. In general, the term "substantially" may be understood as meaning that two or more elements differ by no more than 5% in some way.
It should also be understood that specific values (e.g., in proportions, temperatures, and durations) given herein are not only to be understood as individual values, but are also to be construed as providing endpoints of a range and can be combined with other ranges. Further, particular numerical values given herein are also to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical values set forth herein are approximations that may vary depending upon the requirements.
Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.
In the present application, amino acids may be represented by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission (Biochemical Nomenclature Commission).
In this application, S1、S2And S3Represents a peptide fragment sequence in which amino acid residues are represented using one-letter abbreviations. GLTEGLHGFHVHEFGDNTAGSTSAGPR (SEQ ID No:7) for example is to be understood as representing a peptide stretch consisting of the amino acid residues in the single letter abbreviations.
In the present application, the term "tandem linkage" refers to the linkage of the C-terminus of one peptide chain to the N-terminus of another peptide chain. Optionally, the order of the interconnected peptide chains may be interchanged. For example, the C-terminus of one peptide chain may be linked to the N-terminus of another peptide chain, or the N-terminus of one peptide chain may be linked to the C-terminus of another peptide chain.
In the present application, X1Represents a single arbitrary amino acid residue.
The abbreviation "GLP-1" refers to glucagon-like peptide-1.
The abbreviation "EPO" refers to erythropoietin.
The abbreviation "PKA" refers to protein kinase a.
The abbreviation "EGFR" refers to epidermal growth factor receptor.
The abbreviation "NASH" refers to nonalcoholic steatohepatitis.
With regard to amino acid residues present in the peptide sequences of the present application, L-type residues, i.e. the naturally occurring form, are to be understood, unless otherwise indicated.
The term "functional variant" refers to a variant having the same or similar biological function and properties as the parent. By way of non-limiting example, a "functional variant" may be obtained by making one or more substitutions (e.g., conservative substitutions or D-amino acid residue substitutions) in the parent.
One skilled in the art will recognize that for an alteration, addition or deletion of a single or several amino acid residues in a sequence, a functional variant is considered if the alteration does not affect its original function. For example, an amino acid residue may be substituted with other amino acid residues that are chemically similar, such substitutions being well known in the art. For example, amino acids within the following groups may be substituted for each other, typically as conservative substitutions:
1) alanine (a), glycine (G);
2) aspartic acid (D), glutamic acid (E);
3) asparagine (N), glutamine (Q);
4) arginine (R), lysine (K);
5) isoleucine (I), leucine (L), methionine (M), valine (V);
6) phenylalanine (F), tyrosine (Y), tryptophan (W);
7) serine (S), threonine (T); and
8) cysteine (C), methionine (M).
In some embodiments, a functional variant of a sequence is a variant in which 1 amino acid in the sequence is conservatively substituted with another amino acid.
In some embodiments, a functional variant of a sequence is a variant in which 2 amino acids in the sequence are conservatively substituted with other amino acids.
In some embodiments, a functional variant of a sequence is a variant in which 3 amino acids in the sequence are conservatively substituted with other amino acids.
In some embodiments, a functional variant of a sequence is a variant in which a plurality of amino acids in the sequence are conservatively substituted with other amino acids.
In some embodiments, a functional variant of a sequence is a variant in which 1 amino acid in the sequence is substituted with another amino acid.
In some embodiments, a functional variant of a sequence is a variant in which 2 amino acids in the sequence are substituted with other amino acids.
In some embodiments, a functional variant of a sequence is a variant in which 3 amino acids in the sequence are replaced with other amino acids.
In some embodiments, a functional variant of a sequence is a variant in which a plurality of amino acids in the sequence are substituted with other amino acids.
In some embodiments, a functional variant of a sequence is a variant in which 1 amino acid in the sequence is substituted with the corresponding D-form amino acid.
In some embodiments, a functional variant of a sequence is a variant in which 2 amino acids in the sequence are substituted with the corresponding D-form amino acid.
In some embodiments, a functional variant of a sequence is a variant in which 3 amino acids in the sequence are substituted with the corresponding D-form amino acid.
In some embodiments, a functional variant of a sequence is a variant in which a plurality of amino acids in the sequence are substituted with the corresponding D-form amino acid.
In a first aspect, the present application provides a fusion protein comprising an active protein, a linker and a polypeptide, wherein the polypeptide comprises S1And S2Wherein
S along the direction from N terminal to C terminal1And S2Are connected in series by peptide bonds;
said S1Is an amino acid sequence shown as SEQ ID No. 1 or a functional variant thereof;
said S2Is the amino acid sequence shown as SEQ ID No. 7 or functional variant thereof.
In some embodiments, the polypeptide comprises the structure:
S2-S1or S1-S2
In some embodiments, the structures are shown in order from N-terminus to C-terminus.
In some embodiments, the polypeptide is the amino acid sequence shown in SEQ ID No. 2.
In some embodiments, the polypeptide is prepared by artificial fermentation, and can be obtained by fermentation of Escherichia coli or Pichia yeast, and the fermentation method is known in the art. The polypeptide may be purified using HPLC.
In some embodiments, the fusion protein is a polypeptide prepared by artificial fermentation, and can be obtained by fermentation of Escherichia coli or Pichia yeast, by methods well known in the art. The fusion protein may be purified using HPLC.
In some embodiments, having S2-S1The polypeptides of the structure have a higher self-assembly capacity than those having S1-S2A polypeptide of structure (la).
In some embodiments, having S2-S1The self-assembly ability of a polypeptide of structure is S1-S2About 2-fold greater than the self-assembly capability of the polypeptide of structure.
In some embodiments, the polypeptide comprises S1、S2And S3Wherein
S along the direction from N terminal to C terminal1、S2And S3Are connected in series by peptide bonds;
said S1Is an amino acid sequence shown as SEQ ID No. 1 or a functional variant thereof;
said S2Is an amino acid sequence as shown in SEQ ID No. 7 or a functional variant thereof;
the above-mentionedS3Is the amino acid sequence shown as SEQ ID No. 8 or functional variant thereof.
In some embodiments, the polypeptide comprises the structure:
S3-S2-S1or S3-S1-S2Or S1-S2-S3Or S1-S3-S2Or S2-S1-S3Or S2-S3-S1
In some embodiments, the structures are shown in order from N-terminus to C-terminus.
In some embodiments, the polypeptide is the amino acid sequence shown in SEQ ID No. 3 or SEQ ID No. 4.
In some embodiments, the polypeptide comprises S1、X1And S2Wherein
S along the direction from N terminal to C terminal1、S2、X1Are connected in series by peptide bonds;
said S1Is an amino acid sequence shown as SEQ ID No. 1 or a functional variant thereof;
said S2Is an amino acid sequence as shown in SEQ ID No. 7 or a functional variant thereof;
said X1Is any amino acid.
In some embodiments, said X is1Is lysine or arginine.
In some embodiments, said X is1Is arginine.
In some embodiments, the polypeptide comprises the structure:
S2-X1-S1or S1-X1-S2
In some embodiments, the structures are shown in order from N-terminus to C-terminus.
In some embodiments, the polypeptide is other than X1And S2Comprising in addition two S1。
In some embodiments of the present invention, the substrate is,the polypeptide comprises 2S1、X1And S2。
In some embodiments, the polypeptide comprises the structure:
S2-S1-X1-S1or S1-X1-S1-S2Or S1-S2-X1-S1Or S1-X1-S2-S1Or X1-S1-S2-S1Or S1-S2-S1-X1
In some embodiments, the structures are shown in order from N-terminus to C-terminus.
In some embodiments, the polypeptide is the amino acid sequence shown in SEQ ID No. 5 or SEQ ID No. 6.
In some embodiments, the polypeptide further comprises a pharmaceutically acceptable salt, ester, ether, amide, or mixture thereof.
In some embodiments, the active protein, linker and polypeptide are in that order, in the direction from N-terminus to C-terminus, the active protein, linker and polypeptide.
In some embodiments, the active protein, linker and polypeptide are in that order the polypeptide, linker and active protein in the direction from N-terminus to C-terminus.
In some embodiments, the active protein is a proteinaceous drug.
In some embodiments, the active protein is selected from one or more of the following: GLP-1, EPO, Eryysin A, Exendin-4, PKA competitive polypeptide inhibitors, insulin, monoclonal antibody drugs or fragments thereof.
In some embodiments, the active protein is GLP-1, EPO, Eryysin A, or insulin.
In some embodiments, the active protein is a polypeptide fragment of GLP-1 or a portion thereof.
In some embodiments, the active protein is a polypeptide fragment of EPO or a portion thereof.
In some embodiments, the active protein is a polypeptide fragment of Erylysin a or a portion thereof.
In some embodiments, the active protein is a polypeptide fragment of insulin or a portion thereof.
In some embodiments, a linker is a segment of a polypeptide that serves to separate or link the polypeptide and the active protein. Suitable linkers may be selected based on the following factors: (1) their ability to assume a flexible extended conformation; (2) they do not exhibit secondary structure capable of interacting with functional epitopes on the polypeptide or active protein; and (3) lack of hydrophobic or charged residues that can react with a functional epitope of a polypeptide.
In some embodiments, the linker is about 1-5 amino acids, 5-10 amino acids, 5-25 amino acids, 5-50 amino acids, 10-25 amino acids, 10-50 amino acids, 10-100 amino acids, or any intervening range of amino acids.
In some embodiments, the linker comprises about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids in length. Specific linkers can have about 1-200 amino acids, 1-150 amino acids, 1-100 amino acids, 1-90 amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 1-90 amino acids, 1-4 amino acids, 1-3 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 32, 33, 45, 35, 47, or 2, or more amino acids, 48. 49, 50, 60, 70, 80, 90, 100 or more amino acids in total amino acid length.
The linker may be any one or more of the naturally occurring amino acids, non-naturally occurring amino acids, amino acid analogs, and/or amino acid mimetics as described elsewhere herein and known in the art. Certain amino acid sequences useful as linkers comprise Gly and/or Ser residues. Other near neutral amino acids, such as Thr and Pro, may also be used in the linker if desired.
Certain exemplary linkers include linkers comprising g (gly) and/or s (ser), such as: [ G ]]x、[S]x、[GS]x、[GGS]x、[GSS]x、[GSGS]x、[GGSG]x、[GGGS]x、[GGGGS]xWhereinxIs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more. Other combinations of these and related amino acids will be apparent to those skilled in the art.
In some embodiments, the linker sequence comprises t (thr) and/or p (pro), for example: [ TP ]]xWhereinxIs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more. Other combinations of these and related amino acids will be apparent to those skilled in the art.
In some embodiments, the linker comprises a repeat sequence such as GGGS or TP.
In some embodiments, the linker is a linker as set forth in SEQ ID No. 13.
In some embodiments, the linker is a linker as set forth in SEQ ID No. 14.
In some embodiments, the linker is a linker as set forth in SEQ ID No. 15.
In some embodiments, the linker is a linker as set forth in SEQ ID No: 16.
In some embodiments, the fusion protein is an amino acid sequence as set forth in SEQ ID No. 9.
In some embodiments, the fusion protein is an amino acid sequence as set forth in SEQ ID No. 10.
In some embodiments, the fusion protein is an amino acid sequence as set forth in SEQ ID No. 11.
In some embodiments, the fusion protein is an amino acid sequence as set forth in SEQ ID No. 12.
In a second aspect, the present application provides a pharmaceutical composition comprising the fusion protein of the first aspect and one or more pharmaceutically acceptable excipients.
In some embodiments, the pharmaceutically acceptable excipient is one or more selected from the group consisting of: soluble filler, pH regulator, stabilizer, non-ionic surfactant and osmotic pressure regulator.
In some embodiments, the water soluble filler is one or more selected from the group consisting of: mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose and galactose.
In some embodiments, the pH adjusting agent is one or more selected from the group consisting of: non-volatile acids, physiologically acceptable organic acids, physiologically acceptable inorganic acids, physiologically acceptable bases and salts thereof.
In some embodiments, the pH adjusting agent is one or more selected from the group consisting of: citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid, potassium hydroxide, sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, sodium bicarbonate, potassium bicarbonate, and ammonium bicarbonate.
In some embodiments, the stabilizing agent is one or more selected from the group consisting of: EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, glutamic acid, polyethylene glycol 6000, polyethylene glycol 4000, sodium dodecyl sulfate and tris (hydroxymethyl) aminomethane.
In some embodiments, the stabilizing agent is one or more selected from the group consisting of: sodium metabisulfite, dipotassium hydrogen phosphate, arginine, polyethylene glycol 6000 and tris (hydroxymethyl) aminomethane.
In some embodiments, the nonionic surfactant is a poloxamer.
In some embodiments, the osmolality adjusting agent is one or more selected from the group consisting of: sodium chloride and potassium chloride.
In a third aspect, the present application provides the use of a fusion protein according to the first aspect in the manufacture of a medicament for the treatment of a disease.
In some embodiments, the disease is diabetes, obesity, anemia, or non-alcoholic fatty liver disease.
In some embodiments, the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis.
In a fourth aspect, the present application provides a nucleic acid encoding the fusion protein of the first aspect.
In a fifth aspect, the present application provides an expression system, wherein:
the expression system is an expression vector or expression cassette,
the expression vector or cassette comprises the nucleic acid of the fourth aspect.
In some embodiments, the present application provides an expression vector comprising the nucleic acid of the fourth aspect.
In some embodiments, the present application provides an expression cassette comprising the nucleic acid of the fourth aspect.
In a sixth aspect, the present application provides a cell comprising the fusion protein of the first aspect and/or the nucleic acid of the fourth aspect and/or an expression vector or cassette comprising the nucleic acid of the fourth aspect.
In some embodiments, the present application provides a cell comprising the fusion protein of the first aspect.
In some embodiments, the present application provides a cell comprising a nucleic acid of the fourth aspect.
In some embodiments, the present application provides a cell comprising an expression vector comprising the nucleic acid of the fourth aspect.
In some embodiments, the present application provides a cell comprising an expression cassette comprising a nucleic acid of the fourth aspect.
Examples
The present application is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. Experimental procedures without specific conditions noted in the examples below are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages are by mass and ratios between components are in molar ratios. Unless defined otherwise, all terms of art or science used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are exemplary only.
Experimental materials:
diabetic rats were purchased from the Shanghai SLAC animal center;
the GLP-1EIA kit was purchased from Phoenix Pharmaceuticals, INC;
HFD + CCL4 model animals were purchased from medicy bio-pharmaceutical ltd;
kunming mice were purchased from Shanghai laboratory animals center of Chinese academy of sciences.
Example 1: preparation of fusion protein shown in SEQ ID Nos. 9-12
The fusion proteins referred to in the present application (e.g., SEQ ID Nos: 9-12) can be prepared by fermentation using E.coli as a host cell. The cDNA sequence encoding the fusion protein was ligated into the pET15b plasmid by enzymatic cleavage, followed by transfection of E.coli. The obtained positive clones were subjected to primary fermentation, and then the primary fermentation broth was transferred to a secondary fermentor (500L) and IPTG induction was performed while culturing to OD 2-5. After protein expression, mycelia were collected and cell disruption was performed. And collecting and re-suspending the mycelium crushed liquid, purifying by using a C8 liquid phase column, and freeze-drying for later use.
Example 2: stability of the fusion protein shown as SEQ ID No. 9
Rats with diabetes were injected with GLP-1 (control group) or the fusion protein shown in SEQ ID No:9 (both administered at 0.1M/kg bw), and blood was taken from the ocular plexus vein at different time points about 0.2mL after injection to prepare serum for use.
The blood concentration of GLP-1 in rat serum is detected by adopting an enzyme-linked immunosorbent assay (ELISA), and the operation is as follows: serum was obtained by centrifugation at 4 ℃ and at 13,000rpm/min for 20 minutes. The serum was incubated with 100mM ammonium acetate for 10 minutes at room temperature, and the concentration of GLP-1 was determined using the GLP-1EIA kit. The test method refers to the instruction of the kit and evaluates the stability of GLP-1 according to the result. The results are shown in FIG. 1.
The results show that the fusion protein prolongs the stability of the GLP-1 molecule and reduces the speed of the GLP-1 molecule hydrolyzed by DPP-IV protease.
Example 3: the long-acting hypoglycemic function of the fusion protein shown as SEQ ID No. 10
Rats with diabetes were grouped into groups of 10 rats each, and were orally stimulated with glucose at 2g/kg body weight, followed by oral administration of insulin or fusion protein as shown in SEQ ID No:10 (experimental doses were 0.5mM insulin/kg bw or 0.1mM fusion protein/kg bw, respectively). The blood sugar value of the animals was measured and the results are shown in FIG. 2. The results show that: the oral route of administration of insulin renders insulin rapidly degraded and inactivated in the intestine without drug effect. However, the fusion protein of the present application can protect insulin from the acidic environment or degradation by proteases in the intestine, and the size of the fusion protein meets the requirement of intestinal absorption (60-120 nm).
Example 4: the fusion protein shown as SEQ ID No. 11 has the function of treating NASH
The fusion protein shown as SEQ ID No. 11 is orally administered to HFD + CCL4 model animals, with the administration frequency of 1 time per day and the administration dose of 3mg/kg bw, 15mg/kg bw or 30mg/kg bw. After 4 weeks of administration, the experimental animals were subjected to biochemical marker analysis including measurement of triglyceride, total cholesterol and liver glycogen levels, and the results are shown in Table 1. The results show that the fusion protein shown in SEQ ID No. 11 is capable of treating NASH.
Table 1: the fusion protein shown as SEQ ID No. 11 has the curative effect on HFD + CCL4 model animals (unit: umol/g liver tissue)
Example 5: the fusion protein shown as SEQ ID No. 12 can stimulate the generation of mouse reticulocyte for a long time
Kunming mice were each injected subcutaneously with EPO (once daily at a dose of 0.1mM/kg bw) and the fusion protein shown in SEQ ID No:12 (once every five days at a dose of 0.5mM/kg bw), sacrificed after 42 days, and whole blood was taken for peripheral blood reticulocyte counting and blood cell counting was performed using a fully automatic hemocytometer.
As a result, it was found that the fusion protein shown in SEQ ID No. 12 stimulates an increase in mouse peripheral blood reticulocyte count, indicating that it stimulates erythropoiesis (see Table 2).
Table 2: the fusion protein shown as SEQ ID No. 12 can stimulate the generation of mouse reticulocyte for a long time
Name (R)
Number of reticulocytes
Blank control group
113.68±3.82
Individual EPO groups
805.75±4.67
Fusion protein group
789.76±3.89
Example 6: solubility comparison of the polypeptides shown in SEQ ID Nos 1-4
The four proteins (i.e., the polypeptide shown in SEQ ID No:1, the polypeptide shown in SEQ ID No:2, the polypeptide shown in SEQ ID No:3 and the polypeptide shown in SEQ ID No:4, each 10mg) were dissolved in 1mL of physiological saline, and it was observed by visual observation that the other three proteins (i.e., the polypeptide shown in SEQ ID No:2, the polypeptide shown in SEQ ID No:3 and the polypeptide shown in SEQ ID No: 4) had better solubility than that of SEQ ID No:1, and the observation results are shown in FIG. 3.
Finally, it should be understood that while the various aspects of the present specification describe specific embodiments, those skilled in the art will readily appreciate that the disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Accordingly, it is to be understood that, unless explicitly stated otherwise, the disclosed subject matter is not limited to the particular compositions, methods, and/or formulations, etc., described herein. Moreover, those of ordinary skill in the art will recognize that certain changes, modifications, permutations, variations, additions, subtractions and sub-combinations may be made in accordance with the teachings herein without departing from the spirit of the present specification. It is therefore intended that the following appended claims be interpreted as including all such alterations, modifications, permutations, variations, additions, subtractions and sub-combinations as fall within the true spirit and scope thereof.
Sequence listing
<110> Borui Biotechnology Ltd
<120> fusion protein and use thereof
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 83
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> SITE
<222> (24)..(24)
<223> Xaa is Glu or Lys
<400> 1
Gln Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr
1 5 10 15
Ala Asn Asp Pro Ala Thr Ser Xaa Leu Leu Gly Leu Ile Gly Val Val
20 25 30
Ile Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser
35 40 45
Val Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys
50 55 60
Cys Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro
65 70 75 80
Val Asn Val
<210> 2
<211> 110
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> SITE
<222> (51)..(51)
<223> Xaa is Glu or Lys
<400> 2
Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp
1 5 10 15
Asn Thr Ala Gly Ser Thr Ser Ala Gly Pro Arg Gln Gln Cys Thr Thr
20 25 30
Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr Ala Asn Asp Pro Ala
35 40 45
Thr Ser Xaa Leu Leu Gly Leu Ile Gly Val Val Ile Ser Asp Val Asp
50 55 60
Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser Val Ile Gly Val Gly
65 70 75 80
Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys Cys Asp Ser Ser Pro
85 90 95
Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro Val Asn Val
100 105 110
<210> 3
<211> 149
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Ser Ser Met Ala Thr Lys Ala Val Ser Val Leu Lys Gly Asp Gly Pro
1 5 10 15
Val Gln Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val
20 25 30
Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe
35 40 45
His Val His Glu Phe Gly Asp Asn Thr Ala Gly Ser Thr Ser Ala Gly
50 55 60
Pro Arg Gln Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Glu Ser Thr
65 70 75 80
Ser Thr Ala Asn Asp Pro Ala Thr Ser Glu Leu Leu Gly Leu Ile Gly
85 90 95
Val Val Ile Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro
100 105 110
Ile Ser Val Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro
115 120 125
Val Cys Cys Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys
130 135 140
Val Pro Val Asn Val
145
<210> 4
<211> 149
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Ser Ser Met Ala Thr Lys Ala Val Ser Val Leu Lys Gly Asp Gly Pro
1 5 10 15
Val Gln Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val
20 25 30
Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe
35 40 45
His Val His Glu Phe Gly Asp Asn Thr Ala Gly Ser Thr Ser Ala Gly
50 55 60
Pro Arg Gln Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Glu Ser Thr
65 70 75 80
Ser Thr Ala Asn Asp Pro Ala Thr Ser Lys Leu Leu Gly Leu Ile Gly
85 90 95
Val Val Ile Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro
100 105 110
Ile Ser Val Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro
115 120 125
Val Cys Cys Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys
130 135 140
Val Pro Val Asn Val
145
<210> 5
<211> 194
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp
1 5 10 15
Asn Thr Ala Gly Ser Thr Ser Ala Gly Pro Arg Gln Gln Cys Thr Thr
20 25 30
Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr Ala Asn Asp Pro Ala
35 40 45
Thr Ser Glu Leu Leu Gly Leu Ile Gly Val Val Ile Ser Asp Val Asp
50 55 60
Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser Val Ile Gly Val Gly
65 70 75 80
Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys Cys Asp Ser Ser Pro
85 90 95
Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro Val Asn Val Arg Gln
100 105 110
Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr Ala
115 120 125
Asn Asp Pro Ala Thr Ser Glu Leu Leu Gly Leu Ile Gly Val Val Ile
130 135 140
Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser Val
145 150 155 160
Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys Cys
165 170 175
Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro Val
180 185 190
Asn Val
<210> 6
<211> 194
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp
1 5 10 15
Asn Thr Ala Gly Ser Thr Ser Ala Gly Pro Arg Gln Gln Cys Thr Thr
20 25 30
Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr Ala Asn Asp Pro Ala
35 40 45
Thr Ser Glu Leu Leu Gly Leu Ile Gly Val Val Ile Ser Asp Val Asp
50 55 60
Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser Val Ile Gly Val Gly
65 70 75 80
Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys Cys Asp Ser Ser Pro
85 90 95
Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro Val Asn Val Arg Gln
100 105 110
Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr Ala
115 120 125
Asn Asp Pro Ala Thr Ser Lys Leu Leu Gly Leu Ile Gly Val Val Ile
130 135 140
Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser Val
145 150 155 160
Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys Cys
165 170 175
Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro Val
180 185 190
Asn Val
<210> 7
<211> 27
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp
1 5 10 15
Asn Thr Ala Gly Ser Thr Ser Ala Gly Pro Arg
20 25
<210> 8
<211> 39
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Ser Ser Met Ala Thr Lys Ala Val Ser Val Leu Lys Gly Asp Gly Pro
1 5 10 15
Val Gln Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val
20 25 30
Lys Val Trp Gly Ser Ile Lys
35
<210> 9
<211> 193
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly
20 25 30
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ala Ser Ser Met Ala
35 40 45
Thr Lys Ala Val Ser Val Leu Lys Gly Asp Gly Pro Val Gln Gly Ile
50 55 60
Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp Gly
65 70 75 80
Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu
85 90 95
Phe Gly Asp Asn Thr Ala Gly Ser Thr Ser Ala Gly Pro Arg Gln Gln
100 105 110
Cys Thr Thr Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr Ala Asn
115 120 125
Asp Pro Ala Thr Ser Glu Leu Leu Gly Leu Ile Gly Val Val Ile Ser
130 135 140
Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser Val Ile
145 150 155 160
Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys Cys Asp
165 170 175
Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro Val Asn
180 185 190
Val
<210> 10
<211> 318
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu
1 5 10 15
Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn Gln His Leu Cys Gly
20 25 30
Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe
35 40 45
Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly
50 55 60
Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu
65 70 75 80
Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys
85 90 95
Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn Ser Gly
100 105 110
Thr Pro Thr Pro Thr Pro Thr Pro Thr Gly Glu Phe Gly Leu Thr Glu
115 120 125
Gly Leu His Gly Phe His Val His Glu Phe Gly Asp Asn Thr Ala Gly
130 135 140
Ser Thr Ser Ala Gly Pro Arg Gln Gln Cys Thr Thr Gly Gln Leu Gln
145 150 155 160
Cys Cys Glu Ser Thr Ser Thr Ala Asn Asp Pro Ala Thr Ser Glu Leu
165 170 175
Leu Gly Leu Ile Gly Val Val Ile Ser Asp Val Asp Ala Leu Val Gly
180 185 190
Leu Thr Cys Ser Pro Ile Ser Val Ile Gly Val Gly Ser Gly Ser Ala
195 200 205
Cys Thr Ala Asn Pro Val Cys Cys Asp Ser Ser Pro Ile Gly Gly Leu
210 215 220
Val Ser Ile Gly Cys Val Pro Val Asn Val Arg Gln Gln Cys Thr Thr
225 230 235 240
Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr Ala Asn Asp Pro Ala
245 250 255
Thr Ser Glu Leu Leu Gly Leu Ile Gly Val Val Ile Ser Asp Val Asp
260 265 270
Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser Val Ile Gly Val Gly
275 280 285
Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys Cys Asp Ser Ser Pro
290 295 300
Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro Val Asn Val
305 310 315
<210> 11
<211> 298
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Ala Tyr Ala Gln Trp Val Ile Ile Leu Ile His Asn Val Gly Gln Gln
1 5 10 15
Asn Val Lys Ile Lys Asn Leu Asn Ala Ser Trp Gly Lys Leu Tyr Ala
20 25 30
Asp Gly Asp Lys Asp Thr Glu Val Pro Ala Ser Lys Tyr Glu Gly Met
35 40 45
Val Ile Ala Pro Asp Asp Gln Val Gln Ile Asn Ala Cys Gly Arg Glu
50 55 60
Asp Ala Ala Glu Gly Thr Thr Gly Thr Phe Asp Leu Val Asp Pro Asn
65 70 75 80
Asp Ser Asp Lys Gln Val Arg His Phe Ala Trp Asp Cys Pro Trp Gly
85 90 95
Thr Lys Ala Asn Ser Trp Val Val Gly Gly Ser Asn Ser Lys Trp Met
100 105 110
Ile Glu Tyr Thr Gly Gln Asn Leu Asp Ser Gly Ala Leu Gly Thr Ile
115 120 125
Thr Val Asn Thr Leu Arg Ile Gly Asn Gly Gly Gly Ser Gly Gly Gly
130 135 140
Ser Gly Gly Gly Ser Ser Ser Met Ala Thr Lys Ala Val Ser Val Leu
145 150 155 160
Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe Glu Gln Lys Glu
165 170 175
Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu
180 185 190
Gly Leu His Gly Phe His Val His Glu Phe Gly Asp Asn Thr Ala Gly
195 200 205
Ser Thr Ser Ala Gly Pro Arg Gln Gln Cys Thr Thr Gly Gln Leu Gln
210 215 220
Cys Cys Glu Ser Thr Ser Thr Ala Asn Asp Pro Ala Thr Ser Glu Leu
225 230 235 240
Leu Gly Leu Ile Gly Val Val Ile Ser Asp Val Asp Ala Leu Val Gly
245 250 255
Leu Thr Cys Ser Pro Ile Ser Val Ile Gly Val Gly Ser Gly Ser Ala
260 265 270
Cys Thr Ala Asn Pro Val Cys Cys Asp Ser Ser Pro Ile Gly Gly Leu
275 280 285
Val Ser Ile Gly Cys Val Pro Val Asn Val
290 295
<210> 12
<211> 387
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Ala Pro Pro Pro Asn Leu Pro Asp Pro Lys Phe Glu Ser Lys Ala Ala
1 5 10 15
Leu Leu Ala Ala Arg Gly Pro Glu Glu Leu Leu Cys Phe Thr Glu Arg
20 25 30
Leu Glu Asp Leu Val Cys Phe Trp Glu Glu Ala Ala Ser Ala Gly Val
35 40 45
Gly Pro Gly Asn Tyr Ser Phe Ser Tyr Gln Leu Glu Asp Glu Pro Trp
50 55 60
Lys Leu Cys Arg Leu His Gln Ala Pro Thr Ala Arg Gly Ala Val Arg
65 70 75 80
Phe Trp Cys Ser Leu Pro Thr Ala Asp Thr Ser Ser Phe Val Pro Leu
85 90 95
Glu Leu Arg Val Thr Ala Ala Ser Gly Ala Pro Arg Tyr His Arg Val
100 105 110
Ile His Ile Asn Glu Val Val Leu Leu Asp Ala Pro Val Gly Leu Val
115 120 125
Ala Arg Leu Ala Asp Glu Ser Gly His Val Val Leu Arg Trp Leu Pro
130 135 140
Pro Pro Glu Thr Pro Met Thr Ser His Ile Arg Tyr Glu Val Asp Val
145 150 155 160
Ser Ala Gly Asn Gly Ala Gly Ser Val Gln Arg Val Glu Ile Leu Glu
165 170 175
Gly Arg Thr Glu Cys Val Leu Ser Asn Leu Arg Gly Arg Thr Arg Tyr
180 185 190
Thr Phe Ala Val Arg Ala Arg Met Ala Glu Pro Ser Phe Gly Gly Phe
195 200 205
Trp Ser Ala Trp Ser Glu Pro Val Ser Leu Leu Thr Pro Ser Asp Leu
210 215 220
Asp Pro Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ser Ser
225 230 235 240
Met Ala Thr Lys Ala Val Ser Val Leu Lys Gly Asp Gly Pro Val Gln
245 250 255
Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val
260 265 270
Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val
275 280 285
His Glu Phe Gly Asp Asn Thr Ala Gly Ser Thr Ser Ala Gly Pro Arg
290 295 300
Gln Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr
305 310 315 320
Ala Asn Asp Pro Ala Thr Ser Glu Leu Leu Gly Leu Ile Gly Val Val
325 330 335
Ile Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser
340 345 350
Val Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys
355 360 365
Cys Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro
370 375 380
Val Asn Val
385
<210> 13
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ala
1 5 10
<210> 14
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Ser Gly Thr Pro Thr Pro Thr Pro Thr Pro Thr Gly Glu Phe
1 5 10
<210> 15
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
1 5 10
<210> 16
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Pro Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
1 5 10
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