CN108220315A - The preparation method and fusion protein of a kind of small molecular protein or polypeptide - Google Patents
The preparation method and fusion protein of a kind of small molecular protein or polypeptide Download PDFInfo
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Abstract
The present invention provides the preparation method of a kind of small molecular protein or polypeptide, the method includes：（a）Build engineering strain, the engineering strain includes the recombinant expression carrier with fusion protein nucleic acid sequence encoding, the fusion protein is F L polypeptides, wherein F is fusion partner albumen, selected from aldosterone isomery zymoprotein, aldosterone isomerase protein mutant, human glucagon-like-peptide 1,1 mutant of human glucagon-like-peptide；L is connection peptide, and polypeptide is insulin or its analog, GLP 1 or its analog, GLP 2 or its analog, antibacterial peptide or thymic peptide；（b）The engineering strain is cultivated, to express the fusion protein；With（c）It purifies the fusion protein and passes through protease and cut to obtain the biologically active small molecular protein or polypeptide.The shortcomings that method of the present invention overcomes small molecular protein or polypeptide beyond expression of words, thus albuminoid expression provide new thinking.
Description
Technical field
The present invention relates to the preparation methods of a kind of small molecular protein or polypeptide, further relate to the volume used in the preparation method
Code sequence, recombinant expression carrier and fusion protein.
Background technology
It is shown according to diabetes map (the 7th edition) statistical data that International Diabetes Federation is issued, global glycosuria in 2015
Patient's number has been up to 4.15 hundred million, just has 1 people's illness in average every 11 people, it is contemplated that the year two thousand forty whole world patient of diabetes
Person will increase to 6.42 hundred million.The whole world in 2015 shares 5,000,000 people and dies of diabetes, just there is within average every 6 seconds 1 people death.To each
The illness rate of countries and regions and the estimation of incidence trend show that diabetes mellitus in China patient number increased by 11,200,000 compared with 2013, reached
1.096 hundred million, it ranks the first in the world.According to current development trend, it is contemplated that will be up to 1.507 to the year two thousand forty diabetes mellitus in China patient number
Hundred million.The 12% of Global Health expense is used for treating diabetes and Complication prevention, takes up to 673,000,000,000 dollars within 2015.Cause
This, studies good effect, and cheap Remedies for diabetes is very urgent.
Glucagon-like peptide 1 (GLP-1) be by human glucagon gene code, and by enteron aisle L cells secretion one
Kind peptide hormone, pancreatic beta cell concentration of glucose dependence can be promoted excreting insulin.GLP-1 have GLP-1 (7-37) and
Two kinds of active forms of GLP-1 (7-36) amide, the small peptide being made of respectively 31 amino acid and 30 amino acid.However, people
The natural GLP-1 that body itself generates easily is degraded by internal dipeptidyl peptidase IV (DPP-IV), and plasma half-life divides less than 2
Clock is clinically unable to patent medicine.
The pharmacological action that GLP-1 analogs not only have GLP-1 identical, also with longer plasma half-life, Ke Yixian
Writing reduces blood glucose level and repairing islet cells function.GLP-1 analogs can imitate the drop of people's GLP-1 concentration of glucose dependences
Sugared activity, inhibits patient's gastrointestinal motility and emptying, avoids the deficiency of oral class antidiabetic drug and insulin type antidiabetic drug, as weight increases
Add, long-term blood glucose concentration and HbA1c levels control the adverse reactions such as bad, severe hypoglycemia, significantly improve the life matter of patient
Amount is the important drugs for treating diabetes B.
Exenatide (Exendin-4) is a kind of GLP-1 analogs, is developed by Lilly Co., Eli., is incretin
First member of analog family.Exenatide is isolated from the Monster saliva of North America northwestward, by 39
A amino acid composition has about 53% homology, in mammal body, the physiology of the polypeptide with GLP-1 amino acid sequences
Function is similar to GLP-1, can stimulate the insulin secretion of glucose dependency.Exenatide is unwise to DPP IV
Sense, Half-life in vivo are up to 2-3 hour, have preferable clinical therapeutic efficacy to diabetes B.Clinically it is mainly used for controlling
The type-2 diabetes mellitus of blood glucose cannot be controlled very well by treating melbine, sulfonylurea or melbine and sulfonylurea use in conjunction.
Liraglutide (Liraglutide) is also a kind of GLP-1 analogs, is developed by Novo Nordisk Co., Ltd of Denmark, by 31
A amino acid and a fatty acid side chain composition, amino acid sequence have 97% sequence homology with people GLP-1.With it is natural
GLP-1 is different, and Liraglutide will not be degraded by dipeptidyl peptidase, plasma half-life about 13 hours after subcutaneous administration.Li Lalu
Blood glucose still controls bad patient after peptide is suitable for metformin alone or the treatment of sulfonylurea drugs maximum tolerable dose, with two
First biguanides or sulfonylurea drugs use in conjunction.Daily injection is primary, can inject at any time, without being given according to meal time
Medicine.
Suo Malu peptides (semaglutide) are also a kind of GLP-1 analogs, by 31 amino acid and a fatty acid side chain
Composition, is developed by Novo Nordisk Co., Ltd of Denmark, is to optimize to obtain on the basis of Liraglutide.It uses glutamic acid -2*OEG
Connexon, 18 carbon dicarboxylic acids fatty acid chains, 34 sport arginine in sequence, and 8 alanine mutations are non-natural amino acid
Aib.Three phase clinical research confirmations, the half-life period of Suo Malu peptides is 40h, and far above the 15h of Liraglutide, administration frequency can reach
Weekly.
The preparation method of small molecular protein or polypeptide has two kinds of chemical synthesis and biological synthesis process.Chemical synthesis be with
The amino acid of protection is raw material, connects amino acid one by one successively, then by cracking, purifying, obtains finished product.Biological synthesis process,
Be called DNA recombination methods, by cultivate microbial expression small molecular protein or polypeptide containing target gene, then by separation,
Purifying, obtains finished product.
WO2006119388 is first with Solid phase synthesis GLP-1 analog precursors, then synthesis GLP-1 analogs in the liquid phase；
WO2011006644 is disclosed with four GLP-1 analog segments of solid-state chemical reaction method, then several segments is coupled in the liquid phase
Obtain GLP-1 analogs.It is disclosed using solid-state chemical reaction method in US6902744, CN101538324, CN101357938
The method of GLP-1 analogs.Chinese patent CN102286092, CN102875665 and CN102584982 disclose a kind of GLP-1
The solid phase synthesis process and purification process of analog.
Patent US7595172B2, US2003144471A1 discloses a kind of method for preparing GLP-1 analogs.Including with
Lower key step：1) brewing yeast cell of nucleotide sequence of the culture comprising coding GLP-1 analog precursors, in suitable item
Expression GLP-1 analog precursor molecules under part, 2) the separation GLP-1 analog precursor molecules from zymotic fluid, 3) it is lived with aliphatic acid
Compound is acylated the lysine residue of target peptide, and the lysine residue of N-terminal extension peptide is avoided to be acylated, and 4) it uses
Lysine specificity restriction endonuclease (ALP), which is cut, removes the N section extension peptides of acylated peptide, 5) to detach GLP-1 with suitable mode similar
Object.Patent WO2005019262A1 discloses the preparation method that GLP-1 analog precursors are converted into GLP-1 analogs in detail.
However, saccharomyces cerevisiae easily makes expression product that high glycosylation occur, the risk that immune response occurs for human body is increased.In addition,
Saccharomyces cerevisiae is slow-growing, necessarily causes the production cycle long, objectively increases the cost of production.
Patent CN104894196A discloses a kind of method for preparing GLP-1 analogs, and this method is using hirudin as fusion
Companion, by the splicing of GLP-1 analogs in carrying out amalgamation and expression downstream, and hirudin fusion partner and GLP-1 analogs it
Between design one connection peptide, the connection peptide include TEV enzymes (tobacco etch virus protease) identify cutting sequence, merge egg in this way
GLP-1 analogs can be released by TEV cleavages after white expression.
Patent CN102618552B discloses a kind of method for preparing GLP-1 analogs, and this method GB1 albumen is fusion companion
Companion, by the splicing of GLP-1 analogs in carrying out amalgamation and expression downstream, and one is designed between GB1 albumen and GLP-1 analogs
Peptide is connected, which includes enterokinase cleavage site coded sequence, by expression in escherichia coli fusion protein, Ran Houtong
It crosses enterokinase digestion and releases GLP-1 analogs.
Patent CN103819546A discloses a kind of preparation method of polypeptide lunasine, and this method is with hirudin III (HV3)
For fusion partner, lunasine is connected to the downstream of hirudin III, is connected by connecting peptide GGGDDDDK therebetween, big
Hirudin III- lunasine fusion proteins are expressed in enterobacteria, lunasine is discharged by ox intestine kinase digestion in vitro.
Patent CN100579986C discloses a kind of method for preparing GLP-1 analogs, by 2-32 GLP-1 analog string
Connection expression, and pass through subsequent fusion protein and crack and isolate and purify to obtain GLP-1 analog monomer molecules.But this method table
Up to certain specific amino acid sequences drug molecule when, the C- ends of monomer molecule may remain extra amino acid.
Chemical synthesis, for example, it is desired to using a large amount of organic solvent, be easy to cause environmental pollution there are many defects,
And it is unfavorable for the health of operating personnel, for example, each amino acid extension is required for sufficient washing operation in Solid phase peptide synthssis.
In general, one amino acid of incorporation is related to up to 10 washing steps with this kind of solvent such as NMP, DMF or DCM.In addition, it synthesized
The impurity generated in journey and destination protein property are very close, are difficult removal during purifying, and product yield can not obtain
It is effective to improve, product purity is caused to reduce, influences the quality and drug safety of product.Such as chemical method synthesis GLP-1 classes
During like object, insulin and the like, during synthesis the formation of secondary structure frequently result in each synthesis step efficiency reduce.Cause
This, usually goes out larger peptide or peptide containing certain amino acid sequences with low-purity and produced in yields.Impurity is frequently in finally
The disappearance peptide of one or more amino acid is lost in sequence.These impurity may be very difficult to be separated with required peptide, lead
Product is caused to be polluted by disappearance peptide.Moreover, chemical synthesis needs using protected amino acid and excess reactant is used continuously, make
It is relatively expensive to obtain this method.
Compared with chemical synthesis, biological synthesis process has less pollution, simple for process and of low cost etc.
Advantage.But biological synthesis process is long there are the production cycle at present, the problem of complex process and not high yield.
Invention content
One aspect of the present invention provides the preparation method of a kind of small molecular protein or polypeptide, the method includes：(a) base is built
Because of engineered strain, which includes the recombinant expression carrier with fusion protein nucleic acid sequence encoding, the fusion
Albumen is F-L- polypeptides, and wherein F is fusion partner albumen, selected from aldosterone isomery zymoprotein, aldosterone isomerase protein mutation
Body, human glucagon-like-peptide-1, human glucagon-like-peptide-1 mutant；L is connection peptide, and polypeptide is insulin or its is similar
Object, GLP-1 or its analog, GLP-2 or its analog, antibacterial peptide or thymic peptide；(b) engineering strain is cultivated, with
Express the fusion protein；(c) purify the fusion protein and pass through protease cut to obtain it is biologically active described
Small molecular protein or polypeptide.
In some embodiments, the engineering strain is selected from bacterium, yeast and fungi, preferably bacterium, more
Preferably Escherichia coli (Escherichia coli).
In some embodiments, the connection peptide has below general formula：Xn------X3-X2-X1-X0, wherein, n for 2~
Integer between 8；X0For protease cleavage site, selected from Arg, Lys, preferably Lys；X1And X2Acidic amino acid, i.e. Glu
Or Asp；X3~XnCan be selected from any amino acid in addition to Lys and Arg, preferably His, Glu, Ala, Asp, Gly, Leu,
Val or Met.
In some embodiments, the recombinant expression carrier has T7 promoters or T7lac promoters, preferably T7lac
Promoter.Preferably, the expression vector also additionally has T7 terminators.
In some embodiments, step (c) specifically includes the inclusion body for obtaining the fusion protein, passes through broken and packet
Contain body isolates and purifies to obtain fusion protein crude product, then discharge small molecular protein or polypeptide by protease digestion.For example, one
In a embodiment, the present invention provides a kind of preparation method for recombinating small molecular protein or polypeptide, and this method can obtain Gao Shui
The small molecular protein or polypeptide of flat expression, this method include：(1) recombination of the structure containing the fusion protein nucleic acid sequence encoding
Plasmid；(2) by recombinant plasmid transformed e. coli host bacteria, recombination engineering is obtained；(3) recombination engineering is cultivated, is contained
There is the inclusion body of small molecular protein or polypeptide amalgamation protein；(4) isolate and purify to obtain fusion egg by broken and inclusion body
White crude product, then small molecular protein or polypeptide are discharged by protease digestion.
In some embodiments, step (c) specifically includes the inclusion body for obtaining the fusion protein, passes through broken and packet
Contain body and isolate and purify to obtain fusion protein crude product, then modify by side chain to obtain small molecular protein or more propeptides, finally
It cuts to obtain biologically active small molecular protein or polypeptide by protease.For example, in one embodiment, the present invention
A kind of preparation method for recombinating small molecular protein or polypeptide is provided, this method can obtain high level expression small molecular protein or
Polypeptide, this method include：(1) recombinant plasmid of the structure containing the fusion protein nucleic acid sequence encoding；(2) recombinant plasmid is turned
Change e. coli host bacteria, obtain recombination engineering；(3) recombination engineering is cultivated, obtains and melts containing small molecular protein or polypeptide
The inclusion body of hop protein；(4) small molecular protein or polypeptide amalgamation protein are modified using fatty acid chain activator；(5) by broken
It is broken and inclusion body to isolate and purify to obtain fusion protein crude product, then modify before obtaining small molecular protein or polypeptide by side chain
Body cuts to obtain biologically active small molecular protein or polypeptide finally by protease.
Another aspect of the present invention provides a kind of fusion protein, and general formula is F-L- polypeptides, and wherein F is fusion partner albumen,
Selected from aldosterone isomery zymoprotein, aldosterone isomerase protein mutant, human glucagon-like-peptide-1, human glucagon-like
- 1 mutant of peptide；L for connection peptide, polypeptide for insulin or its analog, GLP-1 or its analog, GLP-2 or its analog,
Antibacterial peptide or thymic peptide.In some embodiments, the nucleic acid sequence encoding of fusion protein is as shown in SEQ ID NO.15.
In some embodiments, the amino acid sequence of fusion protein is as shown in SEQ ID NO.16.
In some embodiments, the aldosterone isomerase protein mutant or human glucagon-like-peptide-1 mutant
To be obtained by corresponding wild-type protein by one or more amino acid mutations, with eliminate specific protein cleavage sites or
Person eliminates side chain decorating site.In some embodiments, it is special to be selected from trypsase, enterokinase, lysine for the protease
Property restriction endonuclease and arginine specificity restriction endonuclease.In some embodiments, the side chain decorating site refers to side chain function
The amino acid residue that group can react with fatty acid chain activator, such as lysine residue.In some embodiments, it merges
The amino acid sequence of companion's aldosterone isomery zymoprotein or its mutant is as shown in SEQ ID NO.13.In some embodiments
In, the amino acid sequence of fusion partner human glucagon-like-peptide-1 or its mutant is as shown in SEQ ID NO.14.
In some embodiments, the connection peptide has below general formula：Xn------X3-X2-X1-X0, wherein, n for 2~
Integer between 8；X0For protease cleavage site, selected from Arg, Lys, preferably Lys；X1And X2Acidic amino acid, i.e. Glu
Or Asp；X3~XnCan be selected from any amino acid in addition to Lys and Arg, preferably His, Glu, Ala, Asp, Gly, Leu,
Val or Met.
In some described modes, the connection peptide is selected from following sequence：
In some embodiments, connection peptide be selected from SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.36 and
SEQ ID NO.43。
Another aspect of the present invention provides a kind of engineering strain, and it includes the volumes with fusion protein of the present invention
The recombinant expression carrier of code nucleic acid sequence.
Another aspect of the present invention provides a kind of recombinant expression carrier, and it includes the cores for encoding fusion protein of the present invention
Acid sequence.
The method of the present invention has many advantages compared with the prior art.For example, the method for the present invention overcomes small molecule
The shortcomings that albumen or polypeptide beyond expression of words, thus albuminoid expression provide new thinking：Small molecular protein or polypeptide are due to dividing
Son is too small, is easily degraded by host strain and is difficult to successful expression.Present invention uses aldosterone isomerase as fusion partner, make
Protein molecular increases 2~5 times, so as to reduce the risk being degraded.The present inventor, which tests, finds a variety of small molecular proteins and more
Peptide, from degradation, realizes correct expression in Escherichia coli.For another example, method destination protein expression quantity of the invention is high：Fusion
Albumen contains the hydrophobic amino acid of higher proportion, and the highly stable inclusion body of structure is easily formed in Escherichia coli, is avoided
The problem of destruction by host protein enzyme.Fusion protein can largely accumulate in cell, obtain higher expression quantity, general feelings
Condition can reach 18~28g/L.In addition, the method growth cycle of the present invention is short, toxigenic capacity is low.Escherichia coli are without special dietary
Ingredient can be grown in simple minimal medium, and the Escherichia coli Growth period is most short in common host strain, generally
25~35h, therefore cost is relatively low.Furthermore the host strain genetic background that the present invention uses understands, safe：Colibacter
Animal derived components are not used in four class bacterium kinds, incubation, harmful virokine will not be introduced.In addition large intestine
The foreign protein degree of glycosylation of bacillus expression is low, and the risk that immune response occurs for human body is low.Moreover, purpose of the present invention albumen
Inclusion body structure is formed, extracting method is simple：Destination protein exists with inclusion bodies, can be simple by broken and centrifugation etc.
Folk prescription method extracts, and inclusion body stable structure, can under freezing conditions store the long period.Finally, it is of the invention to melt
Hop protein can obtain small molecular protein or polypeptide through simple process：Fusion protein is without complex process such as refolding strategies, only
It need to dissolve under suitable conditions, digestion can obtain small molecular protein or polypeptide.Also, due to small molecular protein or polypeptide structure
Simply, correct space structure can be formed in the process, and there is bioactivity.
Description of the drawings
Fig. 1 is the structure diagram of aldosterone isomerase protein mutant expression plasmid pKSI.
Fig. 2 shows the MALDI-TOF-MS analysis results of actrapid monotard's sample.
Fig. 3 shows the SDS-PAGE electrophoresis of engineering bacteria eGLP6 and eGLP8 shaking table culture sample.
Fig. 4 shows the SDS-PAGE electrophoresis of engineering bacteria eGLP6 and eGLP8 fermented and cultured sample.
Fig. 5 shows the SDS-PAGE electrophoresis of engineering bacteria eGLP11 shaking table culture samples.
Fig. 6 shows the SDS-PAGE electrophoresis results of engineering bacteria eGLP12 shaking table culture samples.
The SDS-PAGE electrophoresis results of Fig. 7 engineering bacteria eGLP15 shaking table culture samples.
The SDS-PAGE electrophoresis results of Fig. 8 engineering bacteria eGLP15 fermented and cultured samples.
Fig. 9 shows the RP-HPLC analysis results of GLP-1 analogs.
Figure 10 shows the MALDI-TOF-MS analysis results of GLP-1 analogs.
Figure 11 shows the RP-HPLC analysis results of GLP-1 analogs.
Figure 12 shows the MALDI-TOF-MS analysis results of GLP-1 analogs.
Specific embodiment
The present invention is described further specific examples below is engaged, it is to be understood that the reality gone out given in specification
It applies example to be only exemplary, should not be construed as limiting the scope of the present invention.Those skilled in the art upon review of the specification,
Appropriate change can be made to the embodiment of the present invention, and these changes are still fallen within the scope of the present invention.
In the present invention, when being related to amino acid sequence, letter represents following meanings.
| Chinese | English name | Trigram | Single-letter | Chinese | English name | Trigram | Single-letter |
| Glycine | Glycine | Gly | G | Threonine | Threonine | Thr | T |
| Alanine | Alanine | Ala | A | Cysteine | Cysteine | Cys | C |
| Valine | Valine | Val | v | Methionine | Methionine | Met | M |
| Leucine | Leucine | Leu | L | Asparagine | Asparagine | Asn | N |
| Isoleucine | Isoleucine | Ile | I | Glutamine | Glutamine | Gln | Q |
| Proline | Proline | Pro | P | Tryptophan | Tryptophan | Trp | w |
| Phenylalanine | Phenylalanine | Phe | F | Serine | Serine | Ser | S |
| Tyrosine | Tyrosine | Tyr | Y | Lysine | Lysine | Lys | K |
| Aspartic acid | Aspartic acid | Asp | D | Arginine | Arginine | Arg | R |
| Glutamic acid | Glutamic acid | Glu | E | Histidine | Histidine | His | H |
α-aminoacid (Chinese) 2-methylalanine (English name) Aib (trigram)
The structure of 1 fusion partner expression plasmid of embodiment
Expression plasmid contains T7lac types promoter and T7 terminators, it can be achieved that closing expression during without derivant, there is derivant
In the presence of start expression.Plasmid also contains selected marker, for maintaining stability of the target gene in Escherichia coli.
Using aldosterone isomerase (KSI) be fusion partner albumen, the coding gene sequence of artificial synthesized fusion partner,
5 ' end incorporation initiation codon ATG, the clone of different destination protein molecules, mixes restriction enzyme before initiation codon for convenience
NdeI sites in 3 ' end incorporation restriction enzyme AlwNI sites, are subsequently incorporated into XhoI sites.Synthetic DNA is digested with NdeI and XhoI,
And it is connect with the NdeI-XhoI segments of T7lac type expression plasmids digested.Large intestine of the above-mentioned plasmid in the presence of selection markers
Rise in value in bacillus, detached using conventional method.It is checked and is shown in Plasmid DNA containing insertion sequence by restriction enzyme NdeI and XhoI
Row, sequence analysis show the correct sequence that the Plasmid DNA contains aldosterone isomerase, obtain expression plasmid pKSI (Fig. 1).
Using aldosterone isomerase protein mutant (mKSI) be fusion partner albumen, its artificial synthesized encoding gene sequence
Row using the above method, obtain expression plasmid pmKSI.The use of human glucagon-like-peptide-1 (GLP-1) is fusion partner egg
In vain, its artificial synthesized coding gene sequence using the above method, obtains expression plasmid pGLP.Using human glucagon-like-peptide-
1 mutant (mGLP-1) is fusion partner albumen, its artificial synthesized coding gene sequence using the above method, obtains expression matter
Grain pmGLP.
The structure of 2 escherichia expression system of embodiment and the generation of actrapid monotard
In the suitable connection peptide of aminoterminal addition of human insulin precursor molecule, the mode manually synthesized obtains coding base
Cause, specially L-B (1-29)-AAK-A (1-21), L-B (1-30)-KWK-A (1-21), 3 ' end incorporation terminator codon TAA,
Restriction enzyme XhoI sites are mixed after terminator codon, in 5 ' end incorporation restriction enzyme AlwNI sites.Synthetic DNA with XhoI and
AlwNI digests, and is connect with the XhoI-AlwNI segments of the pKSI plasmids digested.Connection product is imported in Escherichia coli,
And rise in value under ampicillin existence condition, using conventional method separation quality grain.Pass through suitable restriction nuclease enzyme
(such as XhoI, AlwNI, NdeI), which is checked in display Plasmid DNA, contains insetion sequence, and sequence analysis shows that the Plasmid DNA contains
Someone's insulin precursor molecule L-B (1-29)-AAK-A (1-21) (SEQ ID NO.1, SEQ ID NO.3), L-B (1-30)-
The correct sequence of KWK-A (1-21) (SEQ ID NO.2, SEQ ID NO.4).
Recombinant plasmid is transferred in Escherichia coli, in LB tablets (0.5% yeast extract, 1% containing ampicillin
Peptone, 1% sodium chloride, 2% agar powder) on select the E. coli transformant containing recombinant plasmid.Obtain 2 plants of engineering bacterias：
EINS12, eINS16 express aldosterone isomerase-human insulin precursor fusion protein KSI-L-B (1-29)-AAK-A (1- respectively
21)、KSI-L-B(1-30)-KWK-A(1-21)。
Engineering bacteria eINS12, eINS16 are inoculated into LB culture mediums (0.5% yeast extract, 1% peptone, 1% respectively
Sodium chloride), 37 DEG C of 5~8h of culture add in 0.5mM IPTG (isopropyl-β-D-thiogalactoside) and are induced, 28 DEG C of trainings
Support 10h.It after taking 1ml medium centrifugals, collects thalline, adds in 8M urea, ultrasonic disruption (ultrasonic 5s stops 10s, totally 10 minutes)
Afterwards, with SDS-PAGE electrophoretic analysis, it was demonstrated that the presence of fusion protein.The total protein that bacterial cell disruption liquid is measured by lowry methods is dense
Degree measures the concentration of fusion protein with reference to SDS-PAGE results.
Engineering bacteria eINS12 thalline are high-pressure homogeneous broken with 80MPa, and 9000rpm centrifugations obtain inclusion body, molten through 8M urea
Xie Hou obtains desB30 actrapid monotards with lysine specificity endonuclease digestion, connects threonine through transpeptidation reaction, that is, obtains people
Insulin.Transpeptidation reaction is carried out according to this field conventional method, can also be carried out according to the method that patent CN105418755A is provided.
Engineering bacteria eINS16 thalline are high-pressure homogeneous broken with 80MPa, and 9000rpm centrifugations obtain inclusion body, molten through 8M urea
Xie Hou discharges human insulin precursor molecule B (1-30)-K-A (1-21), then through carboxypeptidase with lysine specificity endonuclease digestion
B digestion obtains actrapid monotard.
The actrapid monotard's sample obtained is analyzed with MALDI TOF MS (MALDI-TOF-MS)
It was found that sample mass-to-charge ratio m/z is 5807.355,1 proton molecular weight (1.0073Da) is subtracted, calculating molecular weight analyte is
(5806.35Da theoretical value 5807.69Da).
Fig. 2 shows the MALDI-TOF-MS analysis results of actrapid monotard's sample.
The structure of 3 escherichia expression system of embodiment and fusion protein KSI-L-Arg34The expression of GLP-1 (7-37)
In the suitable connection peptide of aminoterminal addition of GLP-1 analog precursor molecules, the mode manually synthesized is compiled
Code gene, specially L-Arg34GLP-1 (7-37), 3 ' end incorporation terminator codon TAATGA, mixes limit after terminator codon
Enzyme XhoI sites processed, in 5 ' end incorporation restriction enzyme AlwNI sites.Synthetic DNA digests with XhoI and AlwNI, and with digesting
Plasmid pKSI is connected with the XhoI-AlwNI segments of plasmid pmKSI.Connection product is imported in Escherichia coli, and in ammonia benzyl mould
Rise in value under plain existence condition, using conventional method separation quality grain.By suitable restriction nuclease enzyme (such as XhoI,
AlwNI, NdeI) it checks in display Plasmid DNA and contains insetion sequence, it is similar that sequence analysis shows that the Plasmid DNA contains GLP-1
Object precursor molecule L-Arg34The correct sequence (SEQ ID NO.5, SEQ ID NO.6) of GLP-1 (7-37).
Recombinant plasmid is transferred in Escherichia coli, is selected on the LB tablets containing ampicillin containing recombinant plasmid
E. coli transformant.Obtain 2 plants of engineering bacterias：EGLP6 and eGLP8 expresses aldosterone isomerase-GLP-1 analogs and melts respectively
Hop protein KSI-L-Arg34GLP-1 (7-37) and aldosterone isomerase protein mutant-GLP-1 analog fusions mKSI-
L-Arg34GLP-1(7-37)。
Engineering bacteria eGLP6 and eGLP8 are inoculated into LB culture mediums, 37 DEG C of 5~8h of culture add in 0.5mM IPTG (isopropyls
Base-β-D- thiogalactosides) it is induced, 28 DEG C of culture 10h in shaking table.After 1ml medium centrifugals, thalline is collected, is added
Enter 8M urea, after ultrasonic disruption (ultrasonic 5s stops 10s, totally 10 minutes), analyzed for SDS-PAGE, it was demonstrated that fusion protein
In the presence of with Gel-Pro gel analysis softwares calculating fusion protein purity.The total protein of bacterial cell disruption liquid is measured by Lowry methods
Concentration measures the concentration (Fig. 3) of fusion protein with reference to SDS-PAGE results.As a result show that the fusion protein of engineering bacteria eGLP6 is dense
It spends for 0.37g/L, a concentration of 0.48g/L of fusion protein of engineering bacteria eGLP8.
By engineering bacteria eGLP6 and eGLP8 be inoculated into fermentation medium (4g/L glucose, 1mM magnesium sulfate, 0.5g sodium chloride,
1g/L ammonium chlorides, 3g/L potassium dihydrogen phosphates, seven water disodium hydrogen phosphates of 6g/L), 37 DEG C of cultures in 20L fermentation tanks control dissolved oxygen
More than 20%, the glycerite that quality volume fraction is 50% is added after glycerol depletion, is cultivated and is added in IPTG after about 15h and lured
It leads, continues culture about 15h and put tank.According to the method described above with SDS-PAGE characterization fusion proteins (Fig. 4), divided with Gel-Pro gels
It analyses software and calculates fusion protein purity, total protein concentration is measured with Lowry methods.The results show that the fusion protein of engineering bacteria eGLP6
A concentration of 28.2g/L of fusion protein of a concentration of 25.8g/L, engineering bacteria eGLP8.
Fig. 3 shows the SDS-PAGE electrophoresis of engineering bacteria eGLP6 and eGLP8 shaking table culture sample.Wherein swimming lane 1~2
For engineering bacteria eGLP6 (1ml dilutes 5 times)；Swimming lane 3 is Protein standards, and from top to bottom protein molecular weight is respectively
66KD、45KD、35KD、27KD、20KD、14.4KD；Swimming lane 4~5 is engineering bacteria eGLP8 (1ml dilutes 5 times).
Fig. 4 shows the SDS-PAGE electrophoresis of engineering bacteria eGLP6 and eGLP8 fermented and cultured sample.Wherein swimming lane 1 is egg
White matter standard items, from top to bottom protein molecular weight be respectively 66KD, 45KD, 35KD, 27KD, 20KD, 14.4KD, 9.5KD,
6.5KD and 4.1KD；Swimming lane 2 is engineering bacteria eGLP6 (1ml, undiluted)；Swimming lane 3 is engineering bacteria eGLP8 (1ml, undiluted).
The structure of 4 escherichia expression system of embodiment and fusion protein GLP-L-Arg34The generation of GLP-1 (7-37)
With P1 (5 '-atacagatgctggacgatgatgacaaacatgcagaaggca-3 ') and P2 (5 '-
Atactcgagtcattaaccgcggcc-3 ') for primer, with containing L-Arg34The plasmid of GLP-1 (7-37) coded sequence is mould
Plate, PCR amplification obtain GLP-1 analog molecule Ls-R34The encoding gene of GLP-1 (7-37) is digested with XhoI and AlwNI, and with
The XhoI-AlwNI segments connection of the pGLP plasmids digested.Connection product is imported in Escherichia coli, and in ampicillin
Rise in value under existence condition, using conventional method separation quality grain.By suitable restriction nuclease enzyme (such as XhoI,
AlwNI, NdeI) it checks in display Plasmid DNA and contains insetion sequence, it is similar that sequence analysis shows that the Plasmid DNA contains GLP-1
Object precursor molecule L-Arg34The correct sequence (SEQ ID NO.7, SEQ ID NO.8) of GLP-1 (7-37).
With the method in embodiment 3,1 plant of engineering bacteria eGLP11 is obtained, expression human glucagon-like-peptide-GLP-1 is similar
Object fusion protein GLP-L-Arg34GLP-1(7-37).By engineering bacteria eGLP11 in LB medium cultures, fusion protein is a concentration of
0.08g/L。
Fig. 5 shows the SDS-PAGE electrophoresis of engineering bacteria eGLP11 shaking table culture samples.Wherein swimming lane 7 is protein mark
Quasi- product, from top to bottom protein molecular weight be respectively 66KD, 45KD, 35KD, 27KD, 20KD, 14.4KD, 9.5KD, 6.5KD and
4.1KD；Swimming lane 1~3 is that bacteria liquid sample (1ml, undiluted) and swimming lane 4~6 are engineering bacteria before engineering bacteria eGLP11 is induced
Bacteria liquid sample (1ml, undiluted) after eGLP11 inductions.
The structure of 5 escherichia expression system of embodiment and fusion protein KSI-L-Gly8The generation of GLP-1 (7-37)
In the suitable connection peptide of aminoterminal addition of GLP-1 analog precursor molecules, the mode manually synthesized is compiled
Code gene, specially L-Gly8GLP-1 (7-37), 3 ' end incorporation terminator codon TAATGA, mixes limit after terminator codon
Enzyme XhoI sites processed, in 5 ' end incorporation restriction enzyme AlwNI sites.Synthetic DNA digests with XhoI and AlwNI, and with digesting
Plasmid pKSI segments connect.Connection product is imported in Escherichia coli, and is rised in value under ampicillin existence condition, is adopted
With conventional method separation quality grain.It is checked by suitable restriction nuclease enzyme (such as XhoI, AlwNI, NdeI) and shows Plasmid DNA
In contain insetion sequence, sequence analysis shows that the Plasmid DNA contains GLP-1 analog molecule Ls-Gly8GLP-1's (7-37)
Correct sequence (SEQ ID NO.9, SEQ ID NO.10).
Recombinant plasmid is transferred in Escherichia coli, is selected on the LB tablets containing ampicillin containing recombinant plasmid
E. coli transformant.Obtain 1 plant of engineering bacteria eGLP12, expression aldosterone isomerase-GLP-1 analog fusions KSI-
L--Gly8GLP-1(7-37).By engineering bacteria eGLP12 in LB culture medium shaking table cultures, a concentration of 0.41g/L of fusion protein.
Fig. 6 shows the SDS-PAGE electrophoresis results of engineering bacteria eGLP12 shaking table culture samples.Wherein swimming lane 4 is protein
Standard items, from top to bottom protein molecular weight is respectively 66KD, 45KD, 35KD, 27KD, 20KD, 14.4KD, 9.5KD, 6.5KD
And 4.1KD；Swimming lane 1~3 is that bacteria liquid sample (1ml, undiluted) and swimming lane 4~6 are engineering bacteria before engineering bacteria eGLP12 is induced
Bacteria liquid sample (1ml, undiluted) after eGLP12 inductions.
The structure of 6 escherichia expression system of embodiment and fusion protein KSI-L-Arg34The generation of GLP-1 (9-37)
In the suitable connection peptide of aminoterminal addition of GLP-1 analog precursor molecules, the mode manually synthesized is compiled
Code gene, specially L-Arg34GLP-1 (9-37), 3 ' end incorporation terminator codon TAATGA, mixes limit after terminator codon
Enzyme XhoI sites processed, in 5 ' end incorporation restriction enzyme AlwNI sites.Synthetic DNA digests with XhoI and AlwNI, and with digesting
The XhoI-AlwNI segments connection of plasmid pKSI.Connection product is imported in Escherichia coli, and in ampicillin existence condition
Under rise in value, using conventional method separation quality grain.It is examined by suitable restriction nuclease enzyme (such as XhoI, AlwNI, NdeI)
It looks into display Plasmid DNA and contains insetion sequence, sequence analysis shows that the Plasmid DNA contains GLP-1 analog precursor molecules L-
Arg34The correct sequence (SEQ ID NO.11, SEQ ID NO.12) of GLP-1 (9-37).
Recombinant plasmid is transferred in Escherichia coli, is selected on the LB tablets containing ampicillin containing recombinant plasmid
E. coli transformant.Obtain 1 plant of engineering bacteria eGLP15, expression aldosterone isomerase-GLP-1 analog fusions KSI-
L--Arg34GLP-1(9-37)。
With the method in embodiment 3,1 plant of engineering bacteria eGLP15 is obtained, expression aldosterone isomerase-GLP-1 analogs melt
Hop protein GLP-L-Arg34GLP-1(9-37)。
When engineering bacteria eGLP15 is inoculated into LB medium cultures, the expression quantity of fusion protein is 0.33g/L.By engineering bacteria
EGLP10 is inoculated into fermentation medium (4g/L glucose, 1mM magnesium sulfate, 0.5g sodium chloride, 1g/L ammonium chlorides, 3g/L di(2-ethylhexyl)phosphates
Hydrogen potassium, seven water disodium hydrogen phosphates of 6g/L) culture when, the expression quantity of fusion protein is 24.6g/L.Cultural method is the same as embodiment 3.
The SDS-PAGE electrophoresis results of Fig. 7 engineering bacteria eGLP15 shaking table culture samples.Wherein swimming lane 4 is protein standards
Product, from top to bottom protein molecular weight be respectively 66KD, 45KD, 35KD, 27KD, 20KD, 14.4KD, 9.5KD, 6.5KD and
4.1KD；Swimming lane 1~3 is that bacteria liquid sample (1ml, undiluted) and swimming lane 4~6 are engineering bacteria before engineering bacteria eGLP15 is induced
Bacteria liquid sample (1ml, undiluted) after eGLP15 inductions.
The SDS-PAGE electrophoresis results of Fig. 8 engineering bacteria eGLP15 fermented and cultured samples.Wherein swimming lane 1 is protein standards
Product, from top to bottom protein molecular weight be respectively 66KD, 45KD, 35KD, 27KD, 20KD, 14.4KD, 9.5KD, 6.5KD and
4.1KD；Swimming lane 2 induces 14h samples for engineering bacteria eGLP15 (1ml dilutes 20 times)；Swimming lane 3 induces 12h for engineering bacteria eGLP15
Sample (1ml dilutes 20 times)；Swimming lane 3 induces 16h samples for engineering bacteria eGLP15 (1ml dilutes 20 times).
Embodiment 7GLP-1 analogs Arg34Lys26(N- ε-(γ-Glu (N- α-hexadecanoyl group)))-GLP-1 [7-37]
Generation
Take KSI-L-Arg containing fusion protein34The zymotic fluid of GLP-1 (7-37), 9000rpm centrifugations obtain thalline, 80MPa high
Pressure homogeneous crushes to obtain homogenate, and then 9000rpm centrifugations obtain inclusion body.With cleaning solution (100mM Tris, 5mM EDTA,
50mM NaCl, 0.1mM beta -mercaptoethanols, 1%Triton X-100) the primary nucleic acid removed in inclusion body, outer membrane egg will be cleaned
The impurity such as bletilla polysaccharide.Inclusion body is dissolved with 8M urea, is obtained containing aldosterone isomerase-GLP-1 analog precursor fusion eggs
White solution.
The dimethyl sulfoxide (DMSO) (DMSO) of 1.2 times of volumes is added in fusion protein liquid, with n,N-diisopropylethylamine (DIEA)
PH value is adjusted to 10.5-11.5.Every mole of fusion protein add in 5 moles of fatty acids activators (16 phosphinylidyne-Glu (ONHS) of N--
OH), 2-3 hours are stirred to react to get to fusion protein trim KSI-L-Arg34Lys26(N- ε-(γ-Glu (N- α-ten six
Acyl group)))-GLP-1 [7-37].
Every gram of fusion protein adds in the lysine specificity restriction endonuclease of 10AU, with sodium hydroxide and hydrochloric acid tune pH to 7.5~
8.5, room temperature reaction 18~for 24 hours, excision fusion partner (KSI) and connection peptide sequence (L) obtain GLP-1 analogs
Arg34Lys26(N- ε-(γ-Glu (N- α-hexadecanoyl group)))-GLP-1 [7-37].
Endonuclease reaction liquid is taken, Arg is found with high performance liquid chromatography (RP-HPLC) analysis34Lys26-(N-ε-(γ-Glu(N-
α-hexadecanoyl group)))-GLP-1 [7-37] purity be 86.76% (Fig. 9).RP-HPLC effluxes are collected, use ground substance assistant laser
Parsing flight time mass spectrum (MALDI-TOF-MS) analysis finds that sample mass-to-charge ratio m/z is 3753.375, subtracts 1 proton molecule
It measures (1.0073Da), calculates molecular weight analyte as 3752.37Da (theoretical value 3752.20Da) (Figure 10).
Fig. 9 shows the RP-HPLC analysis results of GLP-1 analogs.Wherein peak 3 represents GLP-1 analogs
Arg34Lys26(N- ε-(γ-Glu (N- α-hexadecanoyl group)))-GLP-1 [7-37], purity 86.76%.
Figure 10 shows the MALDI-TOF-MS analysis results of GLP-1 analogs.
Embodiment 8GLP-1 analogs Arg34Lys26(N- ε-(γ-Glu (N- α-hexadecanoyl group)))-GLP-1 [7-37]
Generation
Take mKSI-L-Arg containing fusion protein34The zymotic fluid of GLP-1 (7-37) is handled by method in embodiment 7 and is melted
Hop protein solution.The dimethyl sulfoxide (DMSO) (DMSO) of 1.2 times of volumes is added in fusion protein liquid, uses n,N-diisopropylethylamine
(DIEA) pH value is adjusted to 10.8-11.8.Every mole of fusion protein adds in 2 moles of fatty acids activators (16 phosphinylidyne-Glu of N-
(ONHS)-OH), 2-3 hours are stirred to react to get to fusion protein trim mKSI-L-Arg34Lys26-(N-ε-(γ-Glu
(N- α-hexadecanoyl group)))-GLP-1 [7-37].
With lysine specificity restriction endonuclease excision fusion partner (mKSI) and connect peptide sequence (L) to obtain GLP-1 similar
Object Arg34Lys26(N- ε-(γ-Glu (N- α-hexadecanoyl group)))-GLP-1 [7-37], method is the same as embodiment 7.
Endonuclease reaction liquid is taken, Arg is found with high performance liquid chromatography (RP-HPLC) analysis34Lys26-(N-ε-(γ-Glu(N-
α-hexadecanoyl group)))-GLP-1 [7-37] purity be 83.26% (Figure 11).RP-HPLC effluxes are collected, use ground substance assistant laser
Parsing flight time mass spectrum (MALDI-TOF-MS) analysis finds that sample mass-to-charge ratio m/z is 3752.957, subtracts 1 proton point
Son amount (1.0073Da) calculates molecular weight analyte as 3751.95Da (theoretical value 3752.20Da) (Figure 12).
Figure 11 shows the RP-HPLC analysis results of GLP-1 analogs.Wherein peak 3 represents GLP-1 analogs
Arg34Lys26(N- ε-(γ-Glu (N- α-hexadecanoyl group)))-GLP-1 [7-37], purity 83.26%.
Figure 12 shows the MALDI-TOF-MS analysis results of GLP-1 analogs.
Embodiment 9GLP-1 analogs Gly8The generation of GLP-1 (7-37)
Take KSI-L-Gly containing fusion protein8The zymotic fluid of GLP-1 (7-37), 9000rpm centrifugations obtain thalline, 80MPa high
Pressure homogeneous crushes to obtain homogenate, and then 9000rpm centrifugations obtain thick inclusion body.With cleaning solution (100mM Tris, 5mM
EDTA, 50mM NaCl, 0.1mM beta -mercaptoethanols, 1%Triton X-100) it cleans once, then 9000rpm centrifugations obtain essence
Inclusion body.Smart inclusion body is dissolved with solubilization of inclusion bodies liquid (25mM Tris-HCl, 8M urea, pH8.0), makes fusion protein concentration
For 1.0mg/ml, 2U enterokinase is added in by every milligram of fusion protein, 25 DEG C of reactions are for 24 hours to get to GLP-1 analogs Gly8GLP-
1(7-37)。
Embodiment 10GLP-1 analog N- ε 26- [2- (2- { 2- [2- (2- { 2- [(S) -4- carboxyls -4- (17- carboxyls-ten
Seven alkanoylaminos)-bytyry amino] ethyoxyl } ethyoxyl) acetyl-amino]-ethyoxyl } ethyoxyl)-acetyl group]
[Aib8, Arg34] GLP-1- (7-37) peptide preparation
Take KSI-L-Arg containing fusion protein34The zymotic fluid of GLP-1 (9-37) is handled by the method in embodiment 9, is obtained
GLP-1 analogs Arg34GLP-1(9-37).With acylating reagent 17- { (S) -1- tert-butoxycarbonyls -3- [2- (2- { [2- (2- carboxylics
Ylmethoxy ethyoxyl) ethylaminocarbonyl] methoxyl group } ethyoxyl) ethylaminocarbonyl] propvlcarbamovl } ten
The seven alkanoic acid tert-butyl esters and GLP-1 analogs [Arg34] GLP-1- (9-37) peptide progress acylation reaction, obtain N- ε 26- [2- (2- [2-
(2- [2- (2- [4- (17- tert-butoxycarbonyl heptadecanes sulphonyl-amino) -4 (S)-tert-butoxycarbonyl bytyry amino] ethoxies
Base) ethyoxyl] acetyl-amino) ethyoxyl] ethyoxyl) acetyl group]-[Arg34] GLP-1- (9-37) peptide.Boc-His(Boc)-
Aib-OH and N- ε 26- [2- (2- [2- (2- [2- (2- [4- (17- tert-butoxycarbonyl heptadecanes acyl amino) -4 (S)-tertiary fourth
Epoxide carbonyl bytyry amino] ethyoxyl) ethyoxyl] acetyl-amino)-ethyoxyl] ethyoxyl) acetyl group]-[Arg34]GLP-
1- (9-37) peptide carries out that GLP-1 analog N- ε 26- [2- (2- { 2- [2- (2- { 2- [(S) -4- carboxyls -4- (17- carboxylics are obtained by the reaction
Base-heptadecane acyl amino)-bytyry amino] ethyoxyl } ethyoxyl) acetyl-amino]-ethyoxyl } ethyoxyl)-acetyl
Base] [Aib8, Arg34]GLP-1-(7-37).Experimental method referenced patent CN105154498A is carried out.
SEQUENCE LISTING
<110>Hundred health bio tech ltd of Zhuhai Ji
<120>The preparation method and fusion protein of a kind of small molecular protein or polypeptide
<130> 16617CN
<160> 44
<170> PatentIn version 3.5
<210> 1
<211> 211
<212> DNA
<213>Artificial sequence
<400> 1
aatattcacg catgccagat gctggaagaa aagtttgtta accaacattt gtgtggttct 60
catttggttg aagctttgta cttggtttgt ggtgaaagag gtttcttcta cactccaaag 120
gctgctaagg gtattgttga acaatgttgt acttctattt gttctttgta ccaattggaa 180
aactactgta actaatagct cgagcaccac c 211
<210> 2
<211> 214
<212> DNA
<213>Artificial sequence
<400> 2
aatattcacg catgccagat gctggaagaa aagtttgtta accaacattt gtgtggttct 60
catttggttg aagctttgta cttggtttgt ggtgaaagag gtttcttcta cactccaaag 120
actaagtgga agggtattgt tgaacaatgt tgtacttcta tttgttcttt gtaccaattg 180
gaaaactact gtaactaata gctcgagcac cacc 214
<210> 3
<211> 64
<212> PRT
<213>Artificial sequence
<400> 3
Asn Ile His Ala Cys Gln Met Leu Glu Glu Lys Phe Val Asn Gln His
1 5 10 15
Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu
20 25 30
Arg Gly Phe Phe Tyr Thr Pro Lys Ala Ala Lys Gly Ile Val Glu Gln
35 40 45
Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
50 55 60
<210> 4
<211> 65
<212> PRT
<213>Artificial sequence
<400> 4
Asn Ile His Ala Cys Gln Met Leu Glu Glu Lys Phe Val Asn Gln His
1 5 10 15
Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu
20 25 30
Arg Gly Phe Phe Tyr Thr Pro Lys Thr Lys Trp Lys Gly Ile Val Glu
35 40 45
Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys
50 55 60
Asn
65
<210> 5
<211> 148
<212> DNA
<213>Artificial sequence
<400> 5
aatattcacg catgccagat gctggacgat gacaaacatg cagaaggcac ctttacgagt 60
gatgtgagct cttatctgga aggccaggcg gccaaggaat tcattgcgtg gctggttcgt 120
ggccgcggtt aatgactcga gcaccacc 148
<210> 6
<211> 43
<212> PRT
<213>Artificial sequence
<400> 6
Asn Ile His Ala Cys Gln Met Leu Asp Asp Asp Lys His Ala Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
35 40
<210> 7
<211> 151
<212> DNA
<213>Artificial sequence
<400> 7
aatattcacg catgccagat gctggacgat gatgacaaac atgcagaagg cacctttacg 60
agtgatgtga gctcttatct ggaaggccag gcggccaagg aattcattgc gtggctggtt 120
cgtggccgcg gttaatgact cgagcaccac c 151
<210> 8
<211> 44
<212> PRT
<213>Artificial sequence
<400> 8
Asn Ile His Ala Cys Gln Met Leu Asp Asp Asp Asp Lys His Ala Glu
1 5 10 15
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
20 25 30
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
35 40
<210> 9
<211> 148
<212> DNA
<213>Artificial sequence
<400> 9
aatattcacg catgccagat gctggacgat gacaaacatg gcgaaggcac ctttacgagt 60
gatgtgagct cttatctgga aggccaggcg gccaaggaat tcattgcgtg gctggttcgt 120
ggccgcggtt aatgactcga gcaccacc 148
<210> 10
<211> 43
<212> PRT
<213>Artificial sequence
<400> 10
Asn Ile His Ala Cys Gln Met Leu Asp Asp Asp Lys His Gly Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
35 40
<210> 11
<211> 145
<212> DNA
<213>Artificial sequence
<400> 11
aatattcacg catgccagat gctggatgac gatgacaaag aaggcacctt tacgagtgat 60
gtgagctctt atctggaagg ccaggcggcc aaggaattca ttgcgtggct ggttcgtggc 120
cgcggttaat gactcgagca ccacc 145
<210> 12
<211> 42
<212> PRT
<213>Artificial sequence
<400> 12
Asn Ile His Ala Cys Gln Met Leu Asp Asp Asp Asp Lys Glu Gly Thr
1 5 10 15
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
20 25 30
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
35 40
<210> 13
<211> 125
<212> PRT
<213>Artificial sequence
<220>
<221> MISC_FEATURE
<222> (60, 92, 108, 119)
<223>Xaa=Glu or Asp or Lys or Arg or His, preferably Lys or Arg
<400> 13
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Xaa Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Xaa Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Xaa Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Xaa Asn Ile His Ala Cys Gln
115 120 125
<210> 14
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<221> MISC_FEATURE
<222> (20, 28, 30)
<223>Xaa=Glu or Gln or Asp
<400> 14
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Xaa Gly Xaa
20 25 30
<210> 15
<211> 486
<212> DNA
<213>Artificial sequence
<400> 15
atgcataccc cagaacacat caccgccgtg gtacagcgct ttgtggctgc gctcaatgcc 60
ggcgatctgg acggcatcgt cgcgctgttt gccgatgacg ccacggtgga agaccccgtg 120
ggttccgagc ccaggtccgg tacggctgcg attcgtgagt tttacgccaa ctcgctcaaa 180
ctgcctttgg cggtggagct gacgcaggag gtacgcgcgg tcgccaacga agcggccttc 240
gctttcaccg tcagcttcga gtatcagggc cgcaagaccg tagttgcgcc catcgatcac 300
tttcgcttca atggcgccgg caaggtggtg agcatccgcg ccttgtttgg cgagaagaat 360
attcacgcat gccagatgct ggacgatgac aaacatgcag aaggcacctt tacgagtgat 420
gtgagctctt atctggaagg ccaggcggcc aaggaattca ttgcgtggct ggttcgtggc 480
cgcggt 486
<210> 16
<211> 162
<212> PRT
<213>Artificial sequence
<400> 16
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Asp
115 120 125
Asp Asp Lys His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
130 135 140
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
145 150 155 160
Arg Gly
<210> 17
<211> 7
<212> PRT
<213>Artificial sequence
<400> 17
Gly Gly Gly Gly Glu Glu Lys
1 5
<210> 18
<211> 7
<212> PRT
<213>Artificial sequence
<400> 18
Gly Gly Gly Gly Asp Asp Lys
1 5
<210> 19
<211> 4
<212> PRT
<213>Artificial sequence
<400> 19
Glu Glu Glu Lys
1
<210> 20
<211> 4
<212> PRT
<213>Artificial sequence
<400> 20
Glu Glu Asp Lys
1
<210> 21
<211> 4
<212> PRT
<213>Artificial sequence
<400> 21
Glu Asp Glu Lys
1
<210> 22
<211> 4
<212> PRT
<213>Artificial sequence
<400> 22
Asp Glu Glu Lys
1
<210> 23
<211> 4
<212> PRT
<213>Artificial sequence
<400> 23
Glu Asp Asp Lys
1
<210> 24
<211> 4
<212> PRT
<213>Artificial sequence
<400> 24
Asp Glu Asp Lys
1
<210> 25
<211> 4
<212> PRT
<213>Artificial sequence
<400> 25
Asp Asp Glu Lys
1
<210> 26
<211> 4
<212> PRT
<213>Artificial sequence
<400> 26
Asp Asp Asp Lys
1
<210> 27
<211> 5
<212> PRT
<213>Artificial sequence
<400> 27
Glu Glu Glu Glu Lys
1 5
<210> 28
<211> 5
<212> PRT
<213>Artificial sequence
<400> 28
Glu Glu Glu Asp Lys
1 5
<210> 29
<211> 5
<212> PRT
<213>Artificial sequence
<400> 29
Glu Glu Asp Glu Lys
1 5
<210> 30
<211> 5
<212> PRT
<213>Artificial sequence
<400> 30
Glu Asp Glu Glu Lys
1 5
<210> 31
<211> 5
<212> PRT
<213>Artificial sequence
<400> 31
Asp Glu Glu Glu Lys
1 5
<210> 32
<211> 5
<212> PRT
<213>Artificial sequence
<400> 32
Glu Glu Asp Asp Lys
1 5
<210> 33
<211> 5
<212> PRT
<213>Artificial sequence
<400> 33
Glu Asp Glu Asp Lys
1 5
<210> 34
<211> 5
<212> PRT
<213>Artificial sequence
<400> 34
Asp Glu Glu Asp Lys
1 5
<210> 35
<211> 5
<212> PRT
<213>Artificial sequence
<400> 35
Glu Asp Asp Glu Lys
1 5
<210> 36
<211> 5
<212> PRT
<213>Artificial sequence
<400> 36
Asp Glu Asp Glu Lys
1 5
<210> 37
<211> 5
<212> PRT
<213>Artificial sequence
<400> 37
Asp Asp Glu Glu Lys
1 5
<210> 38
<211> 6
<212> PRT
<213>Artificial sequence
<400> 38
Met Leu Asp Asp Asp Lys
1 5
<210> 39
<211> 5
<212> PRT
<213>Artificial sequence
<400> 39
Asp Glu Asp Asp Lys
1 5
<210> 40
<211> 5
<212> PRT
<213>Artificial sequence
<400> 40
Asp Asp Glu Asp Lys
1 5
<210> 41
<211> 5
<212> PRT
<213>Artificial sequence
<400> 41
Asp Asp Asp Glu Lys
1 5
<210> 42
<211> 5
<212> PRT
<213>Artificial sequence
<400> 42
Asp Asp Asp Asp Lys
1 5
<210> 43
<211> 7
<212> PRT
<213>Artificial sequence
<400> 43
Met Leu Asp Asp Asp Asp Lys
1 5
<210> 44
<211> 7
<212> PRT
<213>Artificial sequence
<400> 44
Met Leu Glu Glu Glu Glu Lys
1 5
Claims (17)
1. the preparation method of a kind of small molecular protein or polypeptide, the method includes：
（a）Engineering strain is built, which includes the recombinant expression with fusion protein nucleic acid sequence encoding
Carrier, the fusion protein are F-L- polypeptides, and wherein F is fusion partner albumen, different selected from aldosterone isomery zymoprotein, aldosterone
Structure zymoprotein mutant, human glucagon-like-peptide-1, human glucagon-like-peptide-1 mutant；L is connection peptide, and polypeptide is pancreas
Island element or its analog, GLP-1 or its analog, GLP-2 or its analog, antibacterial peptide or thymic peptide；
（b）The engineering strain is cultivated, to express the fusion protein；With
（c）It purifies the fusion protein and passes through protease and cut to obtain the biologically active small molecular protein or more
Peptide.
2. according to the method described in claim 1, wherein described engineering strain is selected from bacterium, yeast and fungi.
3. according to the method described in claim 2, wherein described engineering strain is bacterium.
4. according to the method described in claim 2, wherein described engineering strain is Escherichia coli（Escherichia coli）.
5. according to the method described in claim 1, wherein described connection peptide has below general formula：Xn------X3-X2-X1-X0,
In, n is the integer between 2 ~ 8；X0For protease cleavage site, selected from Arg, Lys；X1And X2Acidic amino acid, i.e. Glu or
Asp；X3~XnSelected from any amino acid in addition to Lys and Arg.
6. according to the method described in claim 1, wherein described recombinant expression carrier has T7 promoters or T7 lac promoters.
7. according to the method described in claim 6, wherein described expression vector also additionally has T7 terminators.
8. according to the method described in claim 1, wherein step（c）The inclusion body for obtaining the fusion protein is specifically included, is led to
It crosses and broken and inclusion body isolates and purifies to obtain fusion protein crude product, then small molecular protein or is discharged by protease digestion more
Peptide.
9. according to the method described in claim 1, wherein step（c）The inclusion body for obtaining the fusion protein is specifically included, is led to
It crosses and broken and inclusion body isolates and purifies to obtain fusion protein crude product, then modify to obtain small molecular protein or polypeptide by side chain
Precursor cuts to obtain biologically active small molecular protein or polypeptide finally by protease.
10. a kind of fusion protein, general formula is F-L- polypeptides, and wherein F is fusion partner albumen, selected from aldosterone isomerase egg
In vain, aldosterone isomerase protein mutant, human glucagon-like-peptide-1, human glucagon-like-peptide-1 mutant；L is connection
Peptide, polypeptide are insulin or its analog, GLP-1 or its analog, GLP-2 or its analog, antibacterial peptide or thymic peptide.
11. fusion protein according to claim 10, wherein the aldosterone isomerase protein mutant or the high blood of people's pancreas
Sugared -1 mutant of element sample peptide is to be obtained by corresponding wild-type protein by one or more amino acid mutations, to eliminate specific egg
White cleavage sites eliminate side chain decorating site.
12. fusion protein according to claim 11, wherein fusion partner aldosterone isomery zymoprotein or its mutant
Amino acid sequence is as shown in SEQ ID NO. 13.
13. fusion protein according to claim 11, wherein fusion partner human glucagon-like-peptide-1 or its mutant
Amino acid sequence as shown in SEQ ID NO. 14.
14. fusion protein according to claim 10, wherein the connection peptide has below general formula：Xn------X3-X2-
X1-X0, wherein, n is the integer between 2 ~ 8；X0For protease cleavage site, selected from Arg, Lys；X1And X2It is acidic amino acid,
That is Glu or Asp；X3~XnSelected from any amino acid in addition to Lys and Arg.
15. fusion protein according to claim 10, wherein the connection peptide is selected from SEQ ID NO. 17 to SEQ ID
NO. any one of 46.
16. a kind of engineering strain, it includes the codings with the fusion protein described in claim 10 to 15 any one
The recombinant expression carrier of nucleic acid sequence.
17. a kind of recombinant expression carrier, it includes the nucleic acid of the fusion protein described in coding claim 10 to 15 any one
Sequence.
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| CN111909256A (en) * | 2019-05-10 | 2020-11-10 | 宁波鲲鹏生物科技有限公司 | Polypeptide derivatives and process for preparing the same |
| CN112142852A (en) * | 2020-07-30 | 2020-12-29 | 暨南大学 | Human and mouse egg zona pellucida fusion protein easy to segment and preparation method and application thereof |
| CN112522294A (en) * | 2020-12-24 | 2021-03-19 | 珠海冀百康生物科技有限公司 | Semi-recombinant preparation method of GLP-1 analogue |
| CN112759653A (en) * | 2019-11-06 | 2021-05-07 | 珠海联邦制药股份有限公司 | PKEK fusion protein and preparation method and application thereof |
| CN113201074A (en) * | 2021-05-07 | 2021-08-03 | 珠海联邦制药股份有限公司 | PKEK fusion protein, preparation method and application |
| CN115028740A (en) * | 2022-06-16 | 2022-09-09 | 重庆派金生物科技有限公司 | Method for preparing human parathyroid hormone 1-34 by fusion expression |
| CN115716877A (en) * | 2022-07-04 | 2023-02-28 | 北京惠之衡生物科技有限公司 | Recombinant engineering bacterium for expressing GLP-1 and insulin conjugate polypeptide |
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| CN115716877A (en) * | 2022-07-04 | 2023-02-28 | 北京惠之衡生物科技有限公司 | Recombinant engineering bacterium for expressing GLP-1 and insulin conjugate polypeptide |
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| CN115716877B (en) * | 2022-07-04 | 2023-09-01 | 北京惠之衡生物科技有限公司 | Recombinant engineering bacterium for expressing GLP-1 and insulin conjugate polypeptide |
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