CN110092825A - A method of Liraglutide intermediate polypeptide is prepared using genetic recombination - Google Patents
A method of Liraglutide intermediate polypeptide is prepared using genetic recombination Download PDFInfo
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- CN110092825A CN110092825A CN201910372353.7A CN201910372353A CN110092825A CN 110092825 A CN110092825 A CN 110092825A CN 201910372353 A CN201910372353 A CN 201910372353A CN 110092825 A CN110092825 A CN 110092825A
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Abstract
The invention patent relates to biomedicine fields, are the synthetic methods of the i.e. Arg34-GLP-1 of Liraglutide intermediate polypeptide (7-37) a kind of, include the following steps: to optimize Liraglutide intermediate gene, and construct its recombinant expression carrier；E. coli bl21 is converted, ferments and induces its expressing fusion protein；It is cleaned by sonicated cells, affinity purification, digestion, affinity column excessively later to obtain the higher Liraglutide intermediate of purity.Preparation cumbersome in chemical synthesis, purification step and excessive impurity, the generation of pollutant and longer production cycle are avoided using gene recombination method.Compared with the method for front, production process is simplified, reduces the generation of production cost and pollutant, more conducively large-scale production.
Description
Technical field
The present invention relates to biomedicine field more particularly to a kind of preparation methods of Liraglutide intermediate polypeptide.
Technical background
Diabetes are a kind of common chronic diseases, and symptom includes that secretion of urine is excessive, often has hunger, weight to subtract
Gently, hypopsia etc..The raising of duration blood sugar concentration is also easy to cause the Various Tissues such as heart, blood vessel, eyes, kidney and nerve
Organ lesion influences the health of people.There are 4.25 hundred million diabetics in the whole world within 2017, in All Countries, Chinese glycosuria
Disease burden is most heavy, and patient numbers (1.14 hundred million) account for the whole world nearly 27%.It is expected that 2045, global diabetic will further add
Weight, number is up to 700,000,000.
Human glucagon-like-peptide -1 (GLP-1) analog that Liraglutide (Liraglutide) is, in 2009
European Union's listing, and ratify for treating adult type II diabetes, often in 2011 State Food and Drug Administrations, the China Nian Huo
Day is administered once.It promotes Islet Cells Insulin secretion with concentration of glucose dependent mechanism, plays blood sugar reducing function.Li La
Shandong peptide is currently that Liraglutide precursor molecule is expressed in the way of exocytosis by saccharomyces cerevisiae by Novo Nordisk Co., Ltd
Arg34GLP-1 (7-37), and additional amino acid is cut off after connecting 16 carbon PALM FATTY ACID side chains, obtain Liraglutide molecule.Benefit
Draw Shandong peptide to sell at home as imported medicine, it is expensive so that it is most of it is domestic patient is unbearable must rise, therefore the country is urgent
The a set of Liraglutide manufacture craft of oneself will be developed by being essential, and the price of Liraglutide is greatly lowered, to make
Many patients obtain the effective treatment.
The chemical name of Liraglutide is Arg34Lys26- (N- ε-(y-Glu (N- α-hexadecanoyl group)))-GLP-1, molecule
Formula is C172H265N43O51, molecular weight 3751.20Da, it has 97% homology with natural GLP-1.Liraglutide removes
There is blood sugar reducing function, gastric emptying can also be inhibited, increase satiety, lose weight.
Liraglutide structural formula is as follows:
NH2-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-
Gln-Ala-Ala-Lys-(N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-
Val-Arg-Gly-Arg-Gly-COOH
As shown in the above structural formula, Liraglutide be 34 Lys are become into Arg in natural GLP-1 molecule, and
A 16 carbon PALM FATTY ACID side chains are connected on Lys26, obtain Liraglutide molecule.On the one hand fatty acid side chain can increase benefit
The Reversible binding time of albumin in Shandong peptide and blood is drawn, it is poly- on the other hand can be unified into seven in subcutaneous infusion sites selfing with it
Body delays subcutaneously to absorb it, this two aspect can increase the action time of Liraglutide.
Chemical synthesis process mainly or by amino acid links one by one, and Chinese patent also discloses a series of chemistry
Synthetic method, such as CN105732798, CN102875665, CN102286092 A.For long-chain polypeptide as Liraglutide
For, although can be by solid phase Fully automated synthesis, the production cycle be long, low yield.And by chemical liquid phase subsection synthesis, though
The period of synthesis can be so effectively reduced, but can equally generate and largely be not easy the waste liquid being recycled.
There are two types of obtain benefit as CN108191981A for the bioanalysis synthesis Liraglutide intermediate patent of country's report
The method for drawing Shandong peptide intermediate: first is that the inclusion body after processing ultrasonication, major defect is to use a large amount of urea to be denaturalized,
The annealing efficiency of dilution refolding is very low later and then is unfavorable for digestion and obtains desired polypeptides；Second is that from the supernatant after ultrasonication
Purifying is conducive to subsequent digestion because the albumen of supernatant has natural activity, and the desired polypeptides purity obtained by affinity purification also can
It is relatively very high.If more easy purifying process is selected to be beneficial to further reduce the cost the generation with less contaminants.
Summary of the invention
The present invention provides a kind of method for preparing Liraglutide intermediate polypeptide using genetic recombination, and this method further drops
Low production cost reduces pollutant emission.
The invention adopts the following technical scheme:
A method of Liraglutide intermediate polypeptide is prepared using genetic recombination, comprising the following steps:
(a) Liraglutide intermediate gene is subjected to codon optimization so that it is in E. coli, and
It is building up in expression vector pET-41a, obtains recombinant expression carrier pET-41a-GST-DDDDK- Liraglutide intermediate,
The recombinant protein given expression to is GST-DDDDK- Liraglutide intermediate；
(b) recombinant expression carrier built in step (a) is transformed into e. coli bl21, induces its fermentation with IPTG
Recombinant protein is expressed, precipitating that thalline were collected by centrifugation, sonicated cells after PBS buffer solution is resuspended, centrifugation acquisition supernatant；
(c) it after supernatant liquid filtering being removed thallus, crosses GST affinity purification column and adsorbs recombinant protein GST-DDDDK- Liraglutide
Intermediate is eluted with Tris-HCl elution buffer (reduced glutathione containing 10-40mM) later and collects recombinant protein；
(d) recombinant protein obtained recombination ox intestine kinase (EK) digestion 12-16h；
(e) the mixed liquor tune pH after digestion crosses GST column again and removes foreigh protein removing, and crossing column filtrate is the higher benefit of purity
Draw Shandong peptide intermediate.
Based on the preferred of above-described preparation method, wherein its amino acid sequence such as SEQ ID NO.2 institute in step (a)
Show, the primer sequence of gene order design as shown in SEQ ID NO.1 is as shown in SEQ ID NO.3.
Based on the preferred of above-described preparation method, wherein containing fusion protein encoding gene described in step (a)
Recombinant expression carrier.
Based on the preferred of above-described preparation method, wherein recombinant expression carrier in step (a), among Liraglutide
Body gene fragment clone enters pET-41a carrier and obtains among recombinant expression carrier pET-41a-GST-DDDDK- Liraglutide
Body.
Based on the preferred of above-described preparation method, wherein by label protein GST and ox intestine kinase digestion in step (a)
Site DDDDK genetic fragment is connected to 5 ends ‵ of Liraglutide intermediate gene, the expression vector pET-41a- thus constituted
GST-DDDDK- Liraglutide intermediate.
Based on the preferred of above-described preparation method, wherein by expression vector body pET-41a-GST- in step (a)
DDDDK- Liraglutide intermediate is transferred to e. coli strain bl21, fermentation inducement expressing fusion protein.
Based on the preferred of above-described preparation method, wherein the bacterial strain in step (b) is for obtaining among Liraglutide
Weight histone further obtains Liraglutide intermediate.
Based on the preferred of above-described preparation method, wherein in GST purifying resin fermented liquid supernatant in step (c)
Fusion protein.
Based on the preferred of above-described preparation method, wherein in step (d) with recombination ox intestine kinase to fusion protein into
Row digestion obtains Liraglutide crude intermediate.
Based on the preferred of above-described preparation method, wherein removed among Liraglutide in step (e) by GST resin
Impurity label protein in body crude product obtains the higher Liraglutide intermediate of purity and directly carries out connecting fatty acid side in next step
The reaction of chain.
Using gene recombination method avoid preparation cumbersome in chemical synthesis, purification step and excessive impurity,
The generation of pollutant and longer production cycle.Compared with the method for front, this invention simplifies production processes, reduce life
Produce the generation of cost and pollutant, more conducively large-scale production.
Detailed description of the invention
The structure figures of recombinant plasmid pET-41a-GST-DDDDK- Liraglutide intermediate in Fig. 1 embodiment 1；
Recombinant protein GST-DDDDK- Liraglutide intermediate under being eluted in Fig. 2 embodiment 3；
Specific embodiment
Following embodiment is further elaborated with the present invention, is conventional method unless otherwise specified.
Generally, the present invention provides a kind of method for preparing Liraglutide intermediate polypeptide using genetic recombination, passes through
Construction recombination plasmid pET-41a-GST-DDDDK- Liraglutide intermediate expresses recombinant protein GST- after converting Escherichia coli
DDDDK- Liraglutide intermediate, later by affinity purification, digestion, except the miscellaneous operation acquisition enough Arg34GLP-1 of purity
(7-37), gained crude intermediate can be directly used for subsequent plus fatty acid side chain reaction, to obtain Liraglutide.This method
Production cost is further decreased, pollutant emission is reduced.
The building of 1 recombinant expression carrier of case study on implementation
According to e. coli codon Preference and some other gene order factor relevant to Bacillus coli expression,
Some cis-acting elements are such as avoided, Liraglutide intermediate gene order is optimized and are synthesized.Design is containing homologous later
Its clonal expansion is directed it using one-step cloning kit and is cloned into carrier by the Liraglutide intermediate gene primer of arm
After the DDDDK coded sequence of pET-41a (+) and before terminator sequence, i.e., building obtains recombinant expression carrier pET-41a-GST-
DDDDK- Liraglutide intermediate is as shown in Figure 1.
The building of 2 recombinant strains of case study on implementation
The heat-shock transformed method of obtained recombinant expression carrier routinely is gone into expression type host strain E. coli BL21
(DE3) in.
The expression and purification of 3 recombinant protein of case study on implementation
Obtained recombinant bacterial strain is inoculated into shaking flask culture in the LB culture medium containing final concentration of 50ug/ml ammonia benzyl antibiotic
5h, final concentration of 50ug/mlIPTG induction 4h is added later makes recombinant protein adequate expression.Thalline were collected by centrifugation, is buffered with PBS
After liquid is resuspended, ultrasonication.Supernatant is collected by centrifugation, crosses the affine absorption recombinant protein of GST purification column.It is restored again with containing 20mmol/L
The Tris-HCl buffer of type glutathione elutes lower recombinant protein (about 35kDa) such as Fig. 2.
The purifying of 4 Liraglutide intermediate of case study on implementation
By obtained recombinant protein after ox intestine kinase digestion 16h, by nickel column or GST purification column is affine is adsorbed and removed
Fusion protein GST obtains purer Liraglutide intermediate, and it is anti-that which can be directly used for subsequent plus fatty acid side chain
It answers.
SEQ ID NO.1
ATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATAT
CTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGG
TTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATA
TAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTG
GATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCT
ACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTG
ACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTT
TGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTT
GCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATGGTTCAACTAGTGGTTCTGGTCATC
ACCATCACCATCACTCCGCGGGTCTGGTGCCACGCGGTAGTACTGCAATTGGTATGAAAGAAACCGCTGCTGCTAAA
TTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGGTGGTGGCTCCGGTGATGACGACGACAAGCACGCGGA
GGGTACCTTCACCAGCGATGTGAGCAGCTACCTGGAGGGTCAGGCGGCGAAGGAATTTATCGCGTGGCTGGTTCGTG
GTCGTGGCTAA
SEQ ID NO.2
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSM
AIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGD
HVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGST
SGSGHHHHHHSAGLVPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGDDDDKHAEGTFTSDVSSYLEGQAAKEFI
AWLVRGRG
SEQ ID NO.3
LG-41aF:GATGACGACGACAAGCACGCGGAGGGTACCTTCACC
LG-41aR:GCAAGCTTGTCGACGTTAGCCACGACCACGAACCAG
Claims (10)
1. a kind of method for preparing Liraglutide intermediate polypeptide using genetic recombination, it is characterised in that this method includes following step
It is rapid:
(a) by Liraglutide intermediate gene carry out codon optimization so that it is in E. coli, and by its
It is building up in expression vector pET-41a, obtains recombinant expression carrier pET-41a-GST-DDDDK- Liraglutide intermediate, express
Recombinant protein out is GST-DDDDK- Liraglutide intermediate；
(b) recombinant expression carrier built in step (a) is transformed into e. coli bl21, induces its fermentation expression with IPTG
Recombinant protein, precipitating that thalline were collected by centrifugation, sonicated cells after PBS buffer solution is resuspended, centrifugation obtain supernatant；
(c) it after supernatant liquid filtering being removed thallus, crosses among GST affinity purification column absorption recombinant protein GST-DDDDK- Liraglutide
Body is eluted with Tris-HCl elution buffer (reduced glutathione containing 10-40mM) later and collects recombinant protein；
(d) recombinant protein obtained recombination ox intestine kinase (EK) digestion 12-16h；
(e) the mixed liquor tune pH after digestion crosses GST column again and removes foreigh protein removing, and crossing column filtrate is the higher Li Lalu of purity
Peptide intermediate.
2. preparation method according to claim 1, it is characterised in that: its amino acid sequence such as SEQ ID in step (a)
Shown in NO.2, the primer sequence of gene order design as shown in SEQ ID NO.1 is as shown in SEQ ID NO.3.
3. preparation method according to claim 1, it is characterised in that: encode base containing fusion protein described in step (a)
The recombinant expression carrier of cause.
4. preparation method according to claim 1, it is characterised in that: recombinant expression carrier in step (a), it is characterised in that
Liraglutide intermediate gene fragment clone is entered into pET-41a carrier and obtains recombinant expression carrier pET-41a-GST-DDDDK-
Liraglutide intermediate.
5. preparation method according to claim 1, it is characterised in that: by label protein GST and ox intestine kinase in step (a)
Restriction enzyme site DDDDK genetic fragment is connected to 5 ends ‵ of Liraglutide intermediate gene, the expression vector pET- thus constituted
41a-GST-DDDDK- Liraglutide intermediate.
6. preparation method according to claim 1, it is characterised in that: by expression vector body pET-41a- in step (a)
GST-DDDDK- Liraglutide intermediate is transferred to e. coli strain bl21, fermentation inducement expressing fusion protein.
7. preparation method according to claim 1, it is characterised in that: the bacterial strain in step (b) is for obtaining Liraglutide
Intermediate recombinant protein further obtains Liraglutide intermediate.
8. preparation method according to claim 1, it is characterised in that: use GST purifying resin fermented liquid supernatant in step (c)
In fusion protein.
9. preparation method according to claim 1, it is characterised in that: with recombination ox intestine kinase to fusion egg in step (d)
White carry out digestion, obtains Liraglutide crude intermediate.
10. preparation method according to claim 1, it is characterised in that: remove Li Lalu by GST resin in step (e)
Impurity label protein in peptide crude intermediate obtains the higher Liraglutide intermediate of purity and directly carries out connecting rouge in next step
The reaction of fat acid side chain.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114790473A (en) * | 2021-11-08 | 2022-07-26 | 汉肽生物医药集团有限公司 | Method for in-situ enzyme digestion and purification of liraglutide fusion protein |
| CN116789800A (en) * | 2023-07-12 | 2023-09-22 | 吉林化工学院 | Method for preparing liraglutide bulk drug based on chlorella and liraglutide bulk drug prepared by same |
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| CN116789800A (en) * | 2023-07-12 | 2023-09-22 | 吉林化工学院 | Method for preparing liraglutide bulk drug based on chlorella and liraglutide bulk drug prepared by same |
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