CN110498849A - A kind of main peptide chain of Suo Malu peptide and preparation method thereof - Google Patents
A kind of main peptide chain of Suo Malu peptide and preparation method thereof Download PDFInfo
- Publication number
- CN110498849A CN110498849A CN201910869064.8A CN201910869064A CN110498849A CN 110498849 A CN110498849 A CN 110498849A CN 201910869064 A CN201910869064 A CN 201910869064A CN 110498849 A CN110498849 A CN 110498849A
- Authority
- CN
- China
- Prior art keywords
- peptide
- pet
- suo malu
- glp
- recombinant protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
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Abstract
The invention discloses a kind of main peptide chains of Suo Malu peptide and preparation method thereof, a kind of recombinant protein of Suo Malu peptide intermediate polypeptide of the invention, the recombinant protein of the Suo Malu peptide intermediate polypeptide is leader peptide-DDDDK-GLP-1 (9-37), and the amino acid sequence of the recombinant protein of the Suo Malu peptide intermediate polypeptide is shown in SEQ ID NO.1；In the preparation of Suo Malu peptide intermediate, following leader peptide: (1) MFLKGDGYVQGIINFEGLHHLVALGLV can be used；(2)KSI；(3)TrxA；(4)DsbA,(5)DsbC；(6)Sumo；(7)GST；(8)Intein；(9)KPSTYI.Suo Malu peptide intermediate Purity prepared by the present invention is high, reaches 89% or more, yield is greater than 82%, and the present invention is purified using ionic energy transfer, and separating degree is high, and purification effect is good, and impurity is few, easy to operate.
Description
Technical field
The present invention relates to genetic engineering and polypeptide production methods technical fields, and in particular to a kind of main peptide chain of Suo Malu peptide and
Preparation method.
Background technique
Diabetes are one group of metabolic diseases characterized by hyperglycemia.Hyperglycemia be then due to defect of insulin secretion or
Its biological effect is impaired, or both have concurrently and cause.Long-standing hyperglycemia when diabetes, cause various tissues, especially eye,
Kidney, heart, blood vessel, the chronic lesion of nerve, dysfunction.
Type 1 diabetes, original name insulin-dependent diabetes mellitus mostly occur in Children and teenager, can also betide various
Age.Onset compares sharply, and internal insulin is absolutely insufficient, is easy to happen ketoacidosis, it is necessary to use insulin therapy ability
Satisfactory effect is obtained, otherwise by threat to life.Wherein, type 1 diabetes patient accounts for 10%, and type 2 diabetic patient accounts for 90%.
The long-acting GLP-1 analog that Suo Malu peptide (Semaglutide) is researched and developed by Nove Nordisk company, the drug is only
It needs to carry out subcutaneous administrations once a week, at present also in clinical three stages phase.Structurally, Semaglutide
It needs 26 Lys connecting PEG, glutamic acid and octadecanoid acid aliphatic chain, wherein 8 use unnatural amino acid aminoisobutyric
Acid.Compared with Liraglutide, the aliphatic chain of Semaglutide is longer, and hydrophobicity increases, but Semaglutide is through too short
The PEG of chain is modified, and hydrophily greatly enhances.It can not only combine closely with albumin after PEG modification, cover DPP-4 enzyme hydrolysis
Site, moreover it is possible to reduce renal excretion, biological half-life can be extended, reach macrocyclic effect.
Representative drug one of of the Suo Malu peptide as glucagon peptide (GLP-1) analog, grinds in multiple clinical tests
Studying carefully, which has proved to combine different oral hypoglycemic agents, can effectively control blood glucose, and patient can be made to lose weight, reduce contraction
Pressure and improvement islet beta cell function.
Suo Malu peptide structural formula is as follows:
H-His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-
Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26(PEG-PEG-γ-Glu-
Octadecanedioic Acid)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-
Arg36-Gly37-OH。
From the above structural formula: Suo Malu peptide molecule formula be C187H291N45O59, molecular weight 4113.58, No. CAS
It is that the 34th lysine Lys is changed to arginine Arg on natural GLP-1 molecular structure, wherein 8 for 910463-68-2
Using unnatural amino acid aminoisobutyric acid, and 1 18 carbon fatty acid (PEG-PEG- γ-Glu- is increased at 26
Octadecanedioic Acid) derivative obtained from side chain.
Suo Malu peptide is developed by Novo Nordisk Co., Ltd earliest, by gene recombination technology, is obtained using yeast production.
The synthetic method master of intermediate polypeptide A rg34GLP-1 (9-37) (the 34th amino acids sport Arg by Lys) in the prior art
Chemical synthesis is used, uses a variety of a large amount of organic solvents as patent CN201410784161.4 is disclosed, it is unfriendly to environment,
And complex steps are unfavorable for large-scale industry amplification；Process impurity is more；Total recovery is low.
It is additionally related to disclose expression way in the patent CN104745597A of biological preparation method to be soluble table intracellular
It reaches, expression quantity is lower, is unfavorable for industrialization amplification.It discloses solubilization of inclusion bodies in patent CN104592381A to take long time, body
Product is excessive, uses a large amount of urea；Inclusion body needs long-time renaturation and recombinant protein concentration is low, volume mistake needed for renaturation
Greatly, it is unfavorable for industrial amplification.
The content of present invention prepares Suo Malu using bacillus coli gene recombined engineering the purpose of the present invention is to provide a kind of
The method of the main peptide chain Arg34GLP-1 (9-37) of peptide is obtained by gene recombination technology using Escherichia coli fermentation inducing expression
Leader peptide-DDDDK-Arg34GLP-1 (9-37) recombinant protein, wherein leader peptide of the leader peptide as recombinant protein, facilitates weight
The high level expression of histone and the renaturation of Suo Malu peptide intermediate polypeptide；DDDDK indicates Asp-Asp-asparagus fern
Propylhomoserin-aspartic acid lysine, as enterokinase recognition site, for cutting off the leader peptide of recombinant protein；Arg34GLP-1(9-
37) 9-37 amino acids sequence fragments (the 34th amino acids sport Arg by Lys) of human glucagon-like-peptide are indicated,
It is destination protein sequence.Above-mentioned recombinant protein obtains high yield and high-purity by the operations such as dissolution, refolding strategy, digestion, separation
Polypeptide A rg34GLP-1 (9-37), solve that impurity existing in the prior art is more, yield is low, using a large amount of organic reagents pair
Environment is unfriendly, and solubility expression intracellular causes expression quantity low, and solubilization of inclusion bodies and refolding strategy time are long, and protein concentration is low to be caused
Refolding strategy volume is excessive, is not suitable for large-scale production, and limits the problem of production capacity is promoted.
Currently, lacking a kind of high-purity and main peptide chain of Suo Malu peptide of high yield and preparation method thereof.
Summary of the invention
The purpose of the present invention is in view of the above-mentioned problems, the present invention provides the Suo Malu peptide masters of a kind of high-purity and high yield
Peptide chain and preparation method thereof.
To achieve the goals above, the present invention provides the following technical solution: a kind of Suo Malu peptide intermediate of the invention
The recombinant protein of polypeptide, it is characterised in that: the recombinant protein of the Suo Malu peptide intermediate polypeptide is leader peptide-DDDDK-GLP-
1 (9-37), the amino acid sequence of the recombinant protein of the Suo Malu peptide intermediate polypeptide are shown in SEQ ID NO.1；
In the preparation of Suo Malu peptide intermediate, following leader peptide can be used:
(1)MFLKGDGYVQGIINFEGLHHLVALGLV；(2)KSI；(3)TrxA；(4)DsbA,(5)DsbC；(6)
Sumo；(7)GST；(8)Intein；(9)KPSTYI.
A kind of DNA recombinant expression plasmid of the invention, the encoding gene containing recombinant protein described in coding claim 1.
A kind of recombination engineering comprising the DNA recombinant expression plasmid of the invention, using the genetic recombination
Expression plasmid is transferred to e. coli bl21 (DE3), HMS174 (DE3), JM109 (DE3), T7Express, BL21
(DE3pLyss) it is obtained with any one in Rosetta2 (DE3).
Recombination engineering described in one kind of the invention is in terms of recombinating Suo Malu peptide intermediate GLP-1 (9-37) expression
Using.
A kind of preparation method of main peptide chain of Suo Malu peptide of the invention, it is characterised in that the following steps are included:
(1) composite coding gene, the encoding gene coding recombinant protein；
(2) encoding gene is connected in expression vector, obtains recombinant expression carrier；
(3) recombinant expression carrier with encoding gene is transformed into escherichia coli host, constructs recombination engineering；
(4) recombination engineering containing target gene recombinant expression plasmid is screened using resistant panel；
(5) recombination engineering ferments, and induces the expression of the recombinant protein of insoluble inclusion body form intracellular, the recombination egg
It is white to include amino acid sequence shown in SEQ ID NO.1；
(6) thallus is carried out to high-pressure homogeneous, collection inclusion body, then by inclusion body by washing and refolding strategy；
(7) digestion converts and isolates and purifies to obtain intermediate polypeptide GLP-1 (9-37)；
(8) by intermediate polypeptide and dipeptides (Fmoc-His-Aib-SU) in alkaline aqueous solution, the main peptide of Suo Malu peptide is synthesized
Chain.
Further, in step (2), the DNA recombinant expression plasmid: by cloning the encoding gene
It is weighed into plasmid vector pET-24a (+), pET-28a (+), pET-29a (+), pET-31b (+) or pET-39b (+) are middle
Group expression vector pET-24a (+)-leader peptide-DDDDK-GLP-1 (9-37), pET-28a (+)-leader peptide-DDDDK-GLP-1
(9-37), pET-29a (+)-leader peptide-DDDDK-GLP-1 (9-37), pET-31b (+)-leader peptide-DDDDK-GLP-1 (9-
Or pET-39b (+)-leader peptide-DDDDK-GLP-1 (9-37) 37).
Further, in step (2), the connection type of encoding gene and expression vector be by AlwN I/Xho I,
Hind III/Xho I, Hind III/Nco I, Xho I/Eag I or Sac I/Sal I restriction enzyme site are inserted into plasmid load
Any one corresponding enzyme in body pET-24a (+), pET-28a (+), pET-29a (+), pET-31b (+) or pET-39b (+)
In enzyme site；In the step (5), recombination engineering carries out fermented and cultured, and inducer used in inducing expression is isopropyl sulphur
For galactoside.
Further, in step (6), by the inclusion body after washing in the case where pH is the alkaline condition of 7.5-14, according to
Protein concentration is that solubilization of inclusion bodies buffer is added in 5-40g/L, carries out solubilization of inclusion bodies denaturation.
Further, the digestion conversion, the concrete mode that isolates and purifies are as follows: step (6) refolding strategy in step (7)
PH range of the recombinant protein afterwards through solution described in enterokinase is 10.0-11.0；Recombinant protein warp after step (6) refolding strategy
The mixed liquor of intermediate polypeptide, label and link peptide can be obtained after enterokinase enzymatic hydrolysis 8-12h, mixed liquor uses ion exchange point
From can be obtained the satisfactory intermediate polypeptide sample of purity；.
Further, in step (8), the main peptide chain synthesis reaction solution of Suo Malu peptide is purified water, or is purified water
With the mixed solution of methanol；The pH range of solution is 9.5-11.5.
The utility model has the advantages that Suo Malu peptide intermediate Purity prepared by the present invention is high, reach 89% or more, yield is greater than
82%, the present invention is purified using ionic energy transfer, and separating degree is high, and purification effect is good, and impurity is few, easy to operate.
The present invention compared with the prior art, has the advantage that (1) by changing leader peptide sequences, constructs leader peptide-
DDDDK-GLP-1 (9-37) recombinant protein, expression way become insoluble inclusion body expression intracellular, and expression quantity dramatically increases；(2)
Inclusion body after washing uses alkali soluble, without using a large amount of denaturant, adds according to the high concentration that protein concentration is 30-50g/L
Enter solubilization of inclusion bodies buffer, the refolding strategy time greatly shortens no more than 1h, both can be with digestion, enterokinase enzymolysis time after dissolution
It greatly shortens；Renaturation process is reduced, digestion system is reduced, reduces chemical reagent cost, is conducive to industrialization amplification.
Detailed description of the invention
It is further illustrated below in conjunction with attached drawing, in attached drawing:
The structure figures of recombinant plasmid in Fig. 1 embodiment 1.
3 Suo Malu peptide intermediate peptide T FA-RP-HPLC spectrogram of Fig. 2 embodiment, relative retention time 4.99min.
Fig. 3 Suo Malu peptide intermediate polypeptide MS spectrogram.
The main peptide chain TFA-RP-HPLC spectrogram of Fig. 4 Suo Malu peptide, relative retention time 10.46min.
The main peptide chain polypeptide MS spectrogram of Fig. 5 Suo Malu peptide.
Specific embodiment
For convenient for those skilled in the art understand that the content of present invention, further describes this hair below in conjunction with specific embodiment
Bright technical solution, but the following contents should not in any way limit the claimed range of claims of the present invention.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1
The recombinant protein of a kind of Suo Malu peptide intermediate polypeptide of the invention, it is characterised in that: among the Suo Malu peptide
The recombinant protein of body polypeptide is leader peptide-DDDDK-GLP-1 (9-37), the recombinant protein of the Suo Malu peptide intermediate polypeptide
Amino acid sequence is shown in SEQ ID NO.1；
In the preparation of Suo Malu peptide intermediate, following leader peptide can be used:
(1)MFLKGDGYVQGIINFEGLHHLVALGLV；(2)KSI；(3)TrxA；(4)DsbA,(5)DsbC；(6)
Sumo；(7)GST；(8)Intein；(9)KPSTYI.
A kind of DNA recombinant expression plasmid of the invention, the encoding gene containing recombinant protein described in coding claim 1.
A kind of recombination engineering comprising the DNA recombinant expression plasmid of the invention, using the genetic recombination
Expression plasmid is transferred to e. coli bl21 (DE3), HMS174 (DE3), JM109 (DE3), T7Express, BL21
(DE3pLyss) it is obtained with any one in Rosetta2 (DE3).
Recombination engineering described in one kind of the invention is in terms of recombinating Suo Malu peptide intermediate GLP-1 (9-37) expression
Using.
A kind of preparation method of main peptide chain of Suo Malu peptide of the invention, comprising the following steps:
(1) composite coding gene, the encoding gene coding recombinant protein；
(2) encoding gene is connected in expression vector, obtains recombinant expression carrier；The DNA recombinant expression matter
Grain: by the way that the encoding gene clone is entered plasmid vector pET-24a (+), pET-28a (+), pET-29a (+), pET-
Recombinant expression carrier pET-24a (+)-leader peptide-DDDDK-GLP-1 (9-37) is obtained in 31b (+) or pET-39b (+),
PET-28a (+)-leader peptide-DDDDK-GLP-1 (9-37), pET-29a (+)-leader peptide-DDDDK-GLP-1 (9-37), pET-
31b (+)-leader peptide-DDDDK-GLP-1 (9-37) or pET-39b (+)-leader peptide-DDDDK-GLP-1 (9-37).Encoding gene
Connection type with expression vector is to pass through AlwN I/Xho I, Hind III/Xho I, Hind III/Nco I, Xho I/
Eag I or Sac I/Sal I restriction enzyme site be inserted into plasmid vector pET-24a (+), pET-28a (+), pET-29a (+),
In any one corresponding restriction enzyme site in pET-31b (+) or pET-39b (+).
(3) recombinant expression carrier with encoding gene is transformed into escherichia coli host, constructs recombination engineering；
(4) recombination engineering containing target gene recombinant expression plasmid is screened using resistant panel；
(5) recombination engineering ferments, and induces the expression of the recombinant protein of insoluble inclusion body form intracellular, the recombination egg
It is white to include amino acid sequence shown in SEQ ID NO.1；Recombination engineering carries out fermented and cultured, inducer used in inducing expression
For isopropylthiogalactoside.
(6) thallus is carried out to high-pressure homogeneous, collection inclusion body, then by inclusion body by washing and refolding strategy；It will washing
Inclusion body afterwards is that solubilization of inclusion bodies buffer is added in 40g/L according to protein concentration, carries out in the case where pH is 7.5 alkaline condition
Solubilization of inclusion bodies denaturation.
(7) digestion converts and isolates and purifies to obtain intermediate polypeptide GLP-1 (9-37)；The digestion conversion isolates and purifies
Concrete mode are as follows: the pH range of recombinant protein after step (6) refolding strategy through solution described in enterokinase is 10.0；Step
(6) mixed liquor of intermediate polypeptide, label and link peptide can be obtained in the recombinant protein after refolding strategy after enterokinase digests 8h,
Mixed liquor can be obtained the satisfactory intermediate polypeptide sample of purity using ionic energy transfer.
(8) by intermediate polypeptide and dipeptides (Fmoc-His-Aib-SU) in alkaline aqueous solution, the main peptide of Suo Malu peptide is synthesized
Chain.The main peptide chain synthesis reaction solution of Suo Malu peptide is purified water, and the pH range of solution is 9.5.
Embodiment 2
Embodiment 2 the difference from embodiment 1 is that:
A kind of preparation method of main peptide chain of Suo Malu peptide of the invention, comprising the following steps:
In step (6), thallus is carried out to high-pressure homogeneous, collection inclusion body, inclusion body by washing and is then become multiple
Property；It is that solubilization of inclusion bodies buffering is added in 30g/L according to protein concentration by the inclusion body after washing in the case where pH is 14 alkaline condition
Liquid carries out solubilization of inclusion bodies denaturation.
In step (7), digestion converts and isolates and purifies to obtain intermediate polypeptide GLP-1 (9-37)；The digestion conversion,
The concrete mode isolated and purified are as follows: the recombinant protein after step (6) refolding strategy is through the pH range of solution described in enterokinase
11.0；Intermediate polypeptide, label and connection can be obtained after enterokinase digests 12h in recombinant protein after step (6) refolding strategy
The mixed liquor of peptide, mixed liquor can be obtained the satisfactory intermediate polypeptide sample of purity using ionic energy transfer.
In step (8), by intermediate polypeptide and dipeptides (Fmoc-His-Aib-SU) in alkaline aqueous solution, rope is synthesized
The main peptide chain of Ma Shandong peptide.The main peptide chain synthesis reaction solution of Suo Malu peptide is the mixed solution of purified water and methanol；The pH range of solution
It is 10.
Embodiment 3
Embodiment 3 the difference from embodiment 1 is that:
A kind of preparation method of main peptide chain of Suo Malu peptide of the invention, comprising the following steps:
In step (6), thallus is carried out to high-pressure homogeneous, collection inclusion body, inclusion body by washing and is then become multiple
Property；It is that solubilization of inclusion bodies buffering is added in 5g/L according to protein concentration by the inclusion body after washing in the case where pH is 12 alkaline condition
Liquid carries out solubilization of inclusion bodies denaturation.
In step (7), digestion converts and isolates and purifies to obtain intermediate polypeptide GLP-1 (9-37)；The digestion conversion,
The concrete mode isolated and purified are as follows: the recombinant protein after step (6) refolding strategy is through the pH range of solution described in enterokinase
10.5；Intermediate polypeptide, label and connection can be obtained after enterokinase digests 10h in recombinant protein after step (6) refolding strategy
The mixed liquor of peptide, mixed liquor can be obtained the satisfactory intermediate polypeptide sample of purity using ionic energy transfer.
In step (8), by intermediate polypeptide and dipeptides (Fmoc-His-Aib-SU) in alkaline aqueous solution, rope is synthesized
The main peptide chain of Ma Shandong peptide.The main peptide chain synthesis reaction solution of Suo Malu peptide is purified water, and the pH range of solution is 11.5.
Embodiment 4
The building of recombination engineering
Using the gene of conventional chemical synthesis process synthetic leader peptide-DDDDK-GLP-1 (9-37) recombinant protein, will obtain
The sequence cDNA obtained is inserted into the corresponding restriction enzyme site of plasmid pET-31b (+) by AlwN I/Xho I restriction enzyme site, is built into
Recombinant plasmid as shown in Figure 1, the recombinant plasmid of insertion encoding leader peptide-DDDDK-GLP-1 (9-37) recombinant protein gene is logical
Conventional chemical transformation is crossed to be transferred in host e. coli BL21 (DE3).
Embodiment 5
Engineering bacterium fermentation culture
The resulting recombination engineering positive colony of embodiment 1 is inoculated in LB culture medium, in 37 DEG C of shaken cultivations to OD600
As seed liquor when value reaches 1-2, accesses in the fermentation medium of 7L and cultivated.When fermentation liquid OD600 value reaches 19-21
IPTG is added to be induced, puts tank after inducing 8h.Put after tank that thalline were collected by centrifugation.
Embodiment 6
The purifying of intermediate polypeptide
The resulting fermentation liquid of embodiment 5 put after tank to thalline were collected by centrifugation, broken buffering is added according to w/v 1:7
Inclusion body precipitating is collected by centrifugation by high-pressure homogeneous crusher machine thallus in liquid.Washing is added in precipitating volume ratio 1:15 by weight to delay
Fliud flushing.Solubilization of inclusion bodies buffer is added according to protein concentration for 40g/L in inclusion body after washing, adjusting pH is Alkaline solubilization
1h carries out refolding strategy.Enterokinase is added in dissolved liquid solution of forgiving, and digests after 12h that intermediate can be obtained is more at 37 DEG C
The mixed liquor of peptide, label and link peptide.The mixed liquor for taking the destination protein containing 27.6g, using obtaining purity after ion-exchange purification
For 93.2% intermediate polypeptide sample.Purpose peak sample is taken to carry out HPLC and Mass Spectrometer Method, the destination protein of HPLC detection
Purity is 98.6%, HPLC map as shown in Fig. 2, the destination protein being collected into is 24.1g, yield 87.3%；Mass Spectrometer Method
The molecular weight of destination protein is 3174.86Da, m/z:850.2190 [M+4H] out4+And Suo Malu galanin peptide intermediate molecule amount
Unanimously, as shown in Figure 3.
Embodiment 7
The purifying of intermediate polypeptide
The resulting fermentation liquid of embodiment 5 put after tank to thalline were collected by centrifugation, broken buffering is added according to w/v 1:7
Inclusion body precipitating is collected by centrifugation by high-pressure homogeneous broken thallus in liquid.Washing buffer is added in precipitating volume ratio 1:15 by weight
Liquid, the precipitating being collected by centrifugation after washing are washed 3 times with cleaning solution.Inclusion body after washing is added according to protein concentration for 50g/L
Enter solubilization of inclusion bodies buffer, adjusting pH is that Alkaline solubilization 1h carries out refolding strategy.Enterokinase is added in dissolved liquid solution of forgiving,
The mixed liquor of intermediate polypeptide, label and link peptide can be obtained after enzymatic hydrolysis 7h at 37 DEG C.Take the mixed liquor containing destination protein
Cation exchange chromatography is carried out, eluting peak sample progress HPLC is taken and Mass Spectrometer Method, the purity of HPLC testing goal albumen is
98.8% and the destination protein yield that is collected into be 88.2%；Mass Spectrometer Method goes out the molecular weight and Suo Malu galanin peptide of destination protein
Intermediate molecule amount is consistent.
Embodiment 8
The purifying of intermediate polypeptide
The resulting fermentation liquid of embodiment 5 put after tank to thalline were collected by centrifugation, broken buffering is added according to w/v 1:7
Inclusion body precipitating is collected by centrifugation by high-pressure homogeneous crusher machine thallus in liquid.Washing is added in precipitating volume ratio 1:15 by weight to delay
Fliud flushing.Solubilization of inclusion bodies buffer is added according to protein concentration for 30g/L in inclusion body after washing, adjusting pH is Alkaline solubilization
1h carries out refolding strategy.Enterokinase is added in dissolved liquid solution of forgiving, and digests after 12h that intermediate can be obtained is more at 37 DEG C
The mixed liquor of peptide, label and link peptide.It takes the mixed liquor of the destination protein containing 26.8g to carry out cation exchange chromatography, takes elution
Peak sample carries out HPLC and Mass Spectrometer Method, and the purity of destination protein is 97.9%, and the destination protein yield being collected into is 87.7%；
The molecular weight that Mass Spectrometer Method goes out destination protein is consistent with Suo Malu peptide intermediate molecule amount.
Embodiment 9
The synthesis of the main peptide chain of Suo Malu peptide
The destination protein of collection in embodiment 7 is added in water, is adjusted to pH 10.6 with 1mol/L sodium hydroxide solution,
Stirring makes it completely dissolved.The acetonitrile solution of dipeptides Fmoc-His-Aib-SU is added dropwise, controls entire dropwise addition Process liquor
pH10.5.It is stirred to react 30min, HPLC monitors fully reacting.It is adjusted to 5.0 isoelectric point of pH with acetic acid, there is white precipitate generation,
It is centrifuged to obtain white solid.Decompression is freeze-dried to obtain white Suo Malu peptide crude yield 97%, HPLC purity 98.2%, such as Fig. 4 institute
Show.The molecular weight of destination protein is 3396.84Da, m/z:850.2190 [M+4H]4+And Suo Malu galanin peptide intermediate molecule amount
Unanimously, as shown in Figure 5.
Embodiment 10
The synthesis of the main peptide chain of Suo Malu peptide
The destination protein of collection in embodiment 7 is added in water, is adjusted to pH 10.6 with 1mol/L sodium hydroxide solution,
Stirring makes it completely dissolved.The acetonitrile solution of dipeptides Fmoc-His-Aib-SU is added dropwise, controls entire dropwise addition Process liquor pH
10.0.It is stirred to react 30min, HPLC monitors fully reacting.It is adjusted to 5.0 isoelectric point of pH with acetic acid, there is white precipitate generation, from
Gains in depth of comprehension white solid.Decompression is freeze-dried to obtain white Suo Malu peptide crude yield 97%, HPLC purity 94.95%.Mass Spectrometer Method
The molecular weight of destination protein is consistent with the main peptide chain molecule amount of Suo Malu peptide out.
Embodiment 11
The synthesis of the main peptide chain of Suo Malu peptide
The destination protein of collection in embodiment 7 is added in water, is adjusted to pH 10.6 with 1mol/L sodium hydroxide solution,
Stirring makes it completely dissolved.The acetonitrile solution of dipeptides Fmoc-His-Aib-SU is added dropwise, controls entire dropwise addition Process liquor pH
9.5.It is stirred to react 30min, HPLC monitors fully reacting.It is adjusted to 5.0 isoelectric point of pH with acetic acid, there is white precipitate generation, is centrifuged
Obtain white solid.Decompression is freeze-dried to obtain white Suo Malu peptide crude yield 97%, HPLC purity 92.45%.Mass Spectrometer Method goes out
The molecular weight of destination protein is consistent with the main peptide chain molecule amount of Suo Malu peptide.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, the present invention
Claimed range is delineated by the appended claims, the specification and equivalents thereof from the appended claims.
Sequence table
<110>Nanjing Di Weiao Pharmaceutical Technology Co., Ltd
<120>a kind of main peptide chain of Suo Malu peptide and preparation method thereof
<130> 2019
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> PRT
<213>artificial sequence (amino acid sequence of leader peptide)
<400> 1
Lys Pro Ser Thr Tyr Ile Asp Asp Asp Asp Lys Glu Gly Thr Phe Thr
1 5 10 15
Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Gly Phe Ile
20 25 30
Ala Trp Leu Val Arg Gly Arg Gly
35 40
Claims (10)
1. a kind of recombinant protein of Suo Malu peptide intermediate polypeptide, it is characterised in that: the weight of the Suo Malu peptide intermediate polypeptide
Histone is leader peptide-DDDDK-GLP-1 (9-37), the amino acid sequence of the recombinant protein of the Suo Malu peptide intermediate polypeptide
For shown in SEQ ID NO.1；
In the preparation of Suo Malu peptide intermediate polypeptide, following leader peptide can be used:
(1)MFLKGDGYVQGIINFEGLHHLVALGLV；(2)KSI；(3)TrxA；(4)DsbA,(5)DsbC；(6)
Sumo；(7)GST；(8)Intein；(9)KPSTYI.
2. a kind of DNA recombinant expression plasmid, it is characterised in that: the coding base containing recombinant protein described in coding claim 1
Cause.
3. a kind of recombination engineering comprising DNA recombinant expression plasmid as claimed in claim 2, it is characterised in that: described in use
DNA recombinant expression plasmid be transferred to e. coli bl21 (DE3), HMS174 (DE3), JM109 (DE3), T7Express,
Any one in BL21 (DE3pLyss) and Rosetta2 (DE3) obtains.
4. a kind of recombination engineering as claimed in claim 4 is in terms of recombinating Suo Malu peptide intermediate GLP-1 (9-37) expression
Using.
5. a kind of preparation method of the main peptide chain of Suo Malu peptide, it is characterised in that the following steps are included:
(1) composite coding gene, the encoding gene encode recombinant protein described in claim 1；
(2) encoding gene is connected in expression vector, obtains recombinant expression carrier；
(3) recombinant expression carrier with encoding gene is transformed into escherichia coli host, constructs recombination engineering；
(4) recombination engineering containing target gene recombinant expression plasmid is screened using resistant panel；
(5) recombination engineering ferments, and induces the expression of the recombinant protein of insoluble inclusion body form intracellular, the recombinant protein packet
Amino acid sequence shown in the NO.1 of ID containing SEQ；
(6) thallus is carried out to high-pressure homogeneous, collection inclusion body, then by inclusion body by washing and refolding strategy；
(7) digestion converts and isolates and purifies to obtain intermediate polypeptide GLP-1 (9-37)；
(8) by intermediate polypeptide and dipeptides (Fmoc-His-Aib-SU) in alkaline aqueous solution, the main peptide chain of Suo Malu peptide is synthesized.
6. the preparation method of the main peptide chain of Suo Malu peptide according to claim 5, it is characterised in that: described in step (2)
DNA recombinant expression plasmid: by by the described encoding gene clone enter plasmid vector pET-24a (+), pET-28a (+),
Recombinant expression carrier pET-24a (+)-leader peptide-DDDDK- is obtained in pET-29a (+), pET-31b (+) or pET-39b (+)
GLP-1 (9-37), pET-28a (+)-leader peptide-DDDDK-GLP-1 (9-37), pET-29a (+)-leader peptide-DDDDK-GLP-1
(9-37), pET-31b (+)-leader peptide-DDDDK-GLP-1 (9-37) or pET-39b (+)-leader peptide-DDDDK-GLP-1 (9-
37)。
7. the preparation method of the main peptide chain of Suo Malu peptide according to claim 6, it is characterised in that: in step (2),
The connection type of encoding gene and expression vector is to pass through AlwN I/Xho I, Hind III/Xho I, Hind III/
Nco I, Xho I/Eag I or Sac I/Sal I restriction enzyme site be inserted into plasmid vector pET-24a (+), pET-28a (+),
In any one corresponding restriction enzyme site in pET-29a (+), pET-31b (+) or pET-39b (+)；In the step (5),
Recombination engineering carries out fermented and cultured, and inducer used in inducing expression is isopropylthiogalactoside.
8. according to the preparation method of the main peptide chain of Suo Malu peptide described in claim 6 or 7, it is characterised in that: in step (6)
In, it is that 5-40g/L addition inclusion body is molten according to protein concentration by the inclusion body after washing in the case where pH is the alkaline condition of 7.5-14
Buffer is solved, solubilization of inclusion bodies denaturation is carried out.
9. according to the preparation method of the main peptide chain of Suo Malu peptide described in claim 6 or 7, it is characterised in that: in step (7)
In, it is described digestion conversion, the concrete mode that isolates and purifies are as follows: the recombinant protein after step (6) refolding strategy is through described in enterokinase
The pH range of solution be 10.0-11., the recombinant protein after step (6) refolding strategy can be obtained after enterokinase digests 8-12h
The mixed liquor of intermediate polypeptide, label and link peptide, it is satisfactory that mixed liquor using ionic energy transfer can be obtained purity
Intermediate polypeptide sample.
10. the preparation method of the main peptide chain of Suo Malu peptide according to claim 9, it is characterised in that: in step (8), institute
The main peptide chain synthesis reaction solution of the Suo Malu peptide stated is purified water, or the mixed solution for purified water and methanol；The pH of solution
Range is 9.5-11.5.
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