CN102618552A - Productive technology of recombined exenatide - Google Patents

Abstract

The invention provides a productive technology of recombined exenatide. The productive technology comprises the following steps of: completely deleting a KSI fusion protein sequence in the pET-31b(+) by transforming pET-31b(+), simultaneously deleting a cleavage site of methionine (the cleavage site of ALWN) and a histidine tage (His6-tag) of a C terminal (polypeptide carboxyl terminal) as well as a terminator, replacing by using methionine+His6-tag+GB1 mutation sequence+enterokinase cleavage sequence at the deleting position, and then constructing an engineering bacterium pET-31(b+)-Exendin-4/BL21(DE3) plays. According to the productive technology, the advantage that the pET-31(b+) can produce polypeptides on a larger scale and at high yield is used, promoters with the powerful function of the pET-31(b+) are retained, the shortcoming that a protein product is easy to form an inclusion body in other production technologies is overcome, the N terminal of an objective protein is cut by using the cutting characteristic of the enterokinase, and the cut protein is the Exendin-4 with a natural sequence. The recombined exenatide is obtained through the fermentation expression of the engineering bacterium and the purification of the objective protein. The yield of the recombined exenatide can be up to 500mg/L. Compared with the current most advanced technology, the yield is increased by 20 times, and the recombined exenatide is a soluble protein.

Description

A kind of production technique of the Exenatide of recombinating

Technical field

The invention belongs to the synthesis technical field of biological polypeptide, be specifically related to a kind of production technique of the Exenatide of recombinating.

Background technology

Mellitus are Chronic Non-Communicable Diseasess of another serious harm human health after cardiovascular and cerebrovascular diseases, tumour, in recent years, the change of Along with people's mode of life, diabetes prevalence is rapid ascendant trend.China diabetic subject has exceeded 7,000 ten thousand at present, and diabetic subject's rate of increase is 2 times of American-European countries.(Type 2 Diabetes are the main bodys of mellitus T2DM) to diabetes B, account for diabetic subject's about 90%.T2DM is interacted and is caused the secretion of pancreas islet rope or act on defective by heredity and multiple environmental factors.Its morbidity is higher, is many with the elderly especially, and presents rejuvenation trend.Pancreas islet mouth cells reduced number or secretory functional disturbance are the key links that causes the T2DM morbidity.Treatment is generally followed mode of life intervention (like dietary control, exercise therapy etc.), OHA and is united modes such as using Regular Insulin.The Side effects of pharmaceutical drugs of traditional treatment T2DM are all bigger, and life-time service can excessively cause the MSOF of pancreas islet mouth cells because of insulin secretion.

Glucagon-like peptide 1 (GLP1) and enendin-4 become the focus of treating diabetes research in recent years.Because they have the characteristic that under the blood sugar concentration condition with higher, promotes insulin secretion; And under orthoglycemic situation, do not play a role; Not only can make the diabetes B patient blood sugar recovery of just controlling normal, and sulfonylurea drugs treatment inefficacy person is also had hypoglycemic activity.

Exendin-4 is the straight-chain polypeptide that 39 amino acid is formed, and the saliva separation and Extraction by the huge lizard in South America obtains at first.Its amino-acid residue and Mammals GLP-1 sequence have 53% homology; And the maximum difference of biological effect also almost completely consistent itself and GLP-1 is that N holds dipeptidyl peptidase (DPP-IV) in the 2nd the anti-blood of glycocoll (Gly); The same position of GLP-1 then is the L-Ala (Ala) that is very easily cut off by the DPP-IV, grows (t1/2 =9.7h) so Exendin-4 is absorbed into transformation period behind the blood.Exendin-4 is a kind of strong effect GLP-1 receptor stimulant, can simulate the sugared regulating and controlling effect of this endogenous polypeptide of GLP-1, reduces on an empty stomach and postprandial blood sugar.The activity of Exenatide (Exenatide) is mainly through mediating with human body pancreas GLP-1 receptors bind, by the synthetic and secretion of Regular Insulin that cyclic amp (cAMP) relies on and β mechanism of cell differentiation initiation glucose relies on.Because the amino acid structure of Exendin-4 and biological activity and GLP-1 all have dependency, so also be regarded as a kind of of incretin usually.Because blood sugar reducing function time of Exendin-4 is longer, delay stomach emptying and be different from traditional ofhypoglycemic medicine again with the beneficial effect of impulsion that suppress to ingest to the adiposis patient body weight, so its having a high potential as the exploitation of diabetes B medicine.

The drug research that at present Exendin-4 is carried out is mainly preparation technology, mechanism of action, and the endocrine metabolism disease is clinical, molecular pharmacology and pharmaceutics research.That the Exendin-4 medicine (Exenatide) that has gone on the market adopts is the preparation technology of polypeptide synthesis.

Chemosynthesis cost high (requiring high to raw material amino acid and plant and instrument), output are few; And utilize genetically engineered to produce Exendin-4; Not only can realize industrialized scale operation, reduce production costs effectively, and little to environmental influence, and product quality is safer.But directly express with gene engineering method and to produce 39 such peptides of Exendin-4, expression level is low, and degraded easily, can adopt the method that links amalgamation and expression with carrier proteins (like Trx etc.).But this method also exists the desired polypeptides molecule proportion in the fusion protein molecule very little, and output is very low, and the polypeptide kind is a lot of after the fusion rotein cracking, the problem of separation and purification difficulty.The method of effective increase expression of gene product is to make up the gene concatermer; According to the characteristics of target gene and the characteristic of expression product; Utilization appropriate molecular biologic operation technology is cascaded goal gene by certain mode; Place under the expression vector promotor control and express, again with the concatermer product of expressing with specific material (enzyme or chemical reagent) cracking, with acquisition target peptide monomer.Such as pET-31b (+) is the plasmid of a production polypeptide commonly used; But it is used for producing polypeptide; Design was easily by the intravital proteolyze enzymic hydrolysis of intestinal bacteria for fear of polypeptide originally; So let desired polypeptides and KSI fusion rotein express together, formed inclusion body, this is a very big shortcoming to producing Exendin-4; Can cause the raising greatly of purifying cost and active being difficult to recover if let Exendin-4 generate the inclusion body repeatability earlier, this has been the bottleneck of the industrialization production of reorganization Exenatide.

Summary of the invention

The objective of the invention is to solve the above-mentioned technical problem that exists in the prior art, a kind of production technique of the Exenatide of recombinating is provided.

For realizing above-mentioned purpose, the technical scheme that the present invention adopts is following:

The production technique of reorganization Exenatide of the present invention comprises the structure of engineering bacteria, the fermentation expression of engineering bacteria and the purifying of target protein, and the structure of described engineering bacteria is undertaken by following step:

(1) synthetic of reorganization Exendin-4 polypeptide gene sequence: according to the e. coli codon preferences, the nucleotide sequence of design coding Exendin-4 transform the goal gene shown in SEQ ID No.1 as;

(2) MAG2010 plasmid: get pET31b (+) plasmid; Cut away KSI fusion gene fragment shown in the SEQ ID No.2, be replaced by the gene shown in the SEQ ID No.3, removing restrictions property restriction endonuclease AlwnI restriction enzyme site and methionine(Met) and follow-up sequence; Replacement is with the gene of enteropeptidase restriction enzyme site and target protein; Cut away the histidine mark sequence, add histidine mark, at the two terminators of C-terminal interpolation of target protein in the proteic front of GB1; The plasmid sequence that makes up is: methionine(Met) encoding sequence+6XHis-tag encoding sequence+GB-1 encoding sequence+enteropeptidase restriction enzyme site encoding sequence+with gene+glycocoll coding+two terminators of target protein;

(3) structure of engineering bacteria: the gene fragment that obtains according to the order of the two terminator codons of the cracking site-exedin-4-of promotor-6X Histidine-GB1-tag-enteropeptidase; Utilize the CloneEZ test kit to be connected in the MAG2010 plasmid of well cutting; Obtain recombinant plasmid pET-31 (b+)-Exendin-4, with recombinant plasmid pET-31 (b+)-Exendin-4 with CaCl ₂Method transforms and receives thalline BL21 (DE3) plays, in the LB substratum, is cultivating under 37 ℃, collects thalline, and screening positive clone is as engineering bacteria pET-31 (b+)-Exendin-4/ BL21 (DE3) plays of reorganization Exendin-4 amalgamation and expression.

Make biosynthesizing Exendin-4 can carry out the production application of enlargement of scale, one of crucial is to optimizing fermentation, maximum possible utilize bacterial strain Exendin-4 high yield ability, produce with economic way.

In general; Can not adopt the method for high density fermentation when producing soluble proteins; Because so easy formation inclusion body, the substratum that utilizes is both at home and abroad at present cultivated for 2XYT substratum or LB substratum mostly, utilization be the fask oscillating method cultivation of research in the laboratory mostly.

The fermentation expression of engineering bacteria of the present invention comprises the steps:

(1) choose single bacterium colony to being equipped with in the 30 mL target substratum, 31 ℃, the 200rpm activation is spent the night;

(2) 30mL bacterium liquid is received in the 200 mL 2XYT substratum, 32 ℃, 200rpm cultivates about 2.6 hr;

(3) with 10% inoculum size, receive in the 5 L MMBL substratum, 32 ℃, 240rpm cultivates about 3 hr;

(4) receive in the 60 L fermention mediums;

(5) control growing phase pH is 6.8; Make DO>=10% with stirring velocity and air flow; Treat to begin when the concentration of glucose is reduced to 0.2g/L in the substratum feed supplement; Work as OD ₅₅₀Add 13 mL 1M IPTG when reaching 10 left and right sides and induce, final concentration is 0.2mM, slowly transfers to 7.00 to pH, continues to cultivate after 10 hours and finishes.

The purification step of described target protein adopts the affinity chromatography of ion exchange chromatography-column chromatography-sandwich type to separate and purifying; Engineering bacillus by fermentation, the output of the reorganization Exenatide that obtains of purifying reaches the productive rate of 500 mg/litre.

The affinity chromatography of described sandwich type comprises affinity chromatography one, enzymatic lysis and affinity chromatography two, and concrete steps are following:

(1) affinity chromatography one: adopt the IgG-sepharose-fast flow column of GE company, at first use 50 mM Tris of 3 column volumes, pH 7.5; The damping fluid balance pillar of 150 mM NaCI then with the protein upper prop that will dialyse with same buffer, is washed with 1 times of above-mentioned damping fluid of volume and to be used the 5mM sodium acetate then; The unconjugated impurity of damping fluid flush away of pH5.0 is then used the 500mM sodium acetate, and the damping fluid of pH5.0 elutes target protein; Collect, concentrate; With the 20mM Tris-HCl of capacity, 100mM NaCl, pH7.6 dialysis 12 hours;

(2) enteropeptidase is handled: the recombination ox intestine kinase light chain subunit of 1 μ l is at 20 ℃, 20mM Tris-HCl, 100mM NaCl, 8h cleavage of fusion proteins fully under the condition of pH7.6;

(3) affinity chromatography two: the reorganization Exendin-4 of above-mentioned processing is combined on the nickel post, with initial damping fluid flushing dephosphorylation salt buffer, keeps elution fraction earlier, concentrate lyophilize, the Exenatide that obtains recombinating.

Because Exenatide is a molecular weight ratio smaller polypeptides; And be not suitable for expressing with the system of high molecular weight protein; So we adopt the improved production expression of polypeptides system that is used for specially: pET-31b (+) plasmid and E. Coli (DE3) PlyS produce; Adopting this individual system that following advantage: pET-31b (+) is arranged is a plasmid of producing polypeptide, and desired polypeptides and KSI fusion rotein are expressed together, have formed inclusion body; The polypeptide that can avoid producing is by the intravital proteolyze enzymic hydrolysis of intestinal bacteria; But this is a very big shortcoming to producing Exendin-4, if let Exendin-4 at first generate the form of inclusion body and then raising greatly and active being difficult to that renaturation can cause the purifying cost recover, so we transform pET-31b (+); KSI fusion rotein sequence among the pET-31b (+) is deleted fully, delete methionine(Met) cracking site (ALWN cleavage site) and C-terminal (the polypeptide carboxyl terminal) histidine mark (His simultaneously ₆-tag) and terminator,, replace with methionine(Met)+His in the position of deletion ₆-tag+GB1 mutant nucleotide sequence+enteropeptidase cleavage sequence, the novel plasmid that we form the pET-31b (+) that transforms renames and is Mag2010, and this plasmid has been arranged; We at first obtain the original series of Exendin-4, transform for the preference of genetic codon according to intestinal bacteria then, adopt the genetic sequence that is fit to escherichia coli expression; The complete sequence of the Exendin-4 that synthetic is transformed is cloned then in the PUC57 plasmid of island and is increased on a large scale, with cutting of the Exendin-4 sequence after the amplification and purifying; Be cloned into then in the Mag2010 plasmid of reincarnate; And add dual terminator at C-terminal, the benefit of so doing be utilized pET-31b (+) itself can be extensive, the advantage of the production polypeptide of high yield; The powerful promotor that has kept it; Overcome the shortcoming that this plasmid forms inclusion body easily, utilized the cutting characteristic of enteropeptidase to cut at the N-terminal of target protein, the protein after the cutting is the Exendin-4 with native sequences fully.Utilize the reason of intestinal bacteria E.Coli BL21 (DE3) PlyS expression system to be that Exendin-4 does not need glycosylation modified grade for posttranslational modification; When transcribing, DNA do not need post transcriptional modificaiton yet; So be particularly suitable for producing with Escherichia coli system; Add us and overcome the problem that forms inclusion body, and the advantage of E.Coli BL21 (DE3) PlyS be can mass expressing external albumen, and the interference of toxin such as anti-foreign protein.

The present invention has Exenatide the possibility of 90% formation inclusion body to become 90% formation soluble proteins through connecting GB1 mark fusion rotein.Experiment showed, that the Exenatide that overwhelming majority reorganization produces all is in the soluble protein.

In the system that the Exenatide that at home and abroad carries out is expressed, much human has adopted His ₆-Thioredoxin A (TrxA) fusion rotein and Exendin-4 carry out amalgamation and expression; The fusion protein molecule amount is 14.7+4.2=18.9kd; The ratio of exendin in fusion rotein be 4.2/18.9=22.2%. we adopted less GB1 and the Exendin-4 of self molecular weight to carry out amalgamation and expression; The fusion protein molecule amount is 14.7+4.2=18.9kd, and the ratio of exendin in fusion rotein is 4.2/11.9=35.3%.That is to say that the proportion of Exendin-4 in fusion rotein has increased greatly, under similar system, if the fusion rotein total amount of expressing equates that exendin-4 output in our system is 1.59 times of other systems so.

The present invention adopts the system of GB1 and His-tag double-tagging to make both can adopt nickel post affinity chromatography in purge process, can adopt the GB1 affinity chromatography again, with respect to other purification process, more purifying selection mode can be arranged.

In the existing literature report, output is very low, maximum output of 28 mg/litre just; Utilize fermentation system of the present invention; We can reach the productive rate of 500 mg/litre, and this output is about 20 times of present zymotechnique, so can reduce cost greatly after the large-scale production.Reducing with medicine universal for the medicine cost value of Exenatide class has meaning difficult to the appraisal, and the universal that kind of succeeding in developing afterwards for Regular Insulin just as the recombinant human insulin has important social value and economic worth.

Production technique of the present invention has outstanding feature with respect to present technology: output can reach more than the 500mg/ liter, has improved 20 times than present state-of-the-art process yields, and has been soluble proteins; In process of production, we adopted our company's uniqueness have independent intellectual property right zymotechnique, output improves greatly; The dual affinity purification technology that we adopt; Cooperate traditional purification process, purification efficiency is greatly improved, the purifying of thick extraction in early stage; Alleviate the pressure of follow-up purifying, reduced the cost of purifying.

Description of drawings

Growth curve of bacteria figure in Fig. 1 fermenting process.

Fig. 2 is Fermentation Process of Parameter monitoring result figure.

Fig. 3 is the stability analysis figure as a result of plasmid.

Fig. 4 is that the cell of plasmid includes spirogram.

Fig. 5 is the detected result figure of active cells.

Fig. 6 is polyacrylamide gel electrophoresis figure.

Embodiment

Below in conjunction with embodiment the present invention is elaborated.

One, the structure of engineering bacteria

(1) synthetic of reorganization Exendin-4 polypeptide gene sequence: according to the e. coli codon preferences, the nucleotide sequence of design coding Exendin-4 transform the goal gene shown in SEQ ID No.1 as; Gene clone after synthetic is advanced among the plasmid PUC57, transforms among the bacillus coli DH 5 а to increase, and the plasmid extraction kit with EZ Bioresearch company carries out purifying then, and it is subsequent use that the goal gene after the cutting goes out purifying.

(2) MAG2010 plasmid: MAG2010 plasmid: get pET31b (+) plasmid; Cut away KSI fusion gene fragment shown in the SEQ ID No.2, be replaced by the gene shown in the SEQ ID No.3, removing restrictions property restriction endonuclease AlwnI restriction enzyme site and methionine(Met) and follow-up sequence; Replacement is with the gene of enteropeptidase restriction enzyme site and target protein; Cut away the histidine mark sequence, add histidine mark, at the two terminators of C-terminal interpolation of target protein in the proteic front of GB1; The plasmid sequence that makes up is: methionine(Met) encoding sequence+6XHis-tag encoding sequence+GB-1 encoding sequence+enteropeptidase restriction enzyme site encoding sequence+with gene+glycocoll coding+two terminators of target protein.

(3) structure of engineering bacteria: the gene fragment that obtains according to the order of the two terminator codons of the cracking site-exedin-4-of promotor-6X Histidine-GB1-tag-enteropeptidase; Utilize the CloneEZ test kit to be connected in the MAG2010 plasmid of well cutting; Obtain recombinant plasmid pET-31 (b+)-Exendin-4, with recombinant plasmid pET-31 (b+)-Exendin-4 with CaCl ₂Method transforms and receives thalline BL21 (DE3) plays, in the LB substratum, is cultivating under 37 ℃, collects thalline, and screening positive clone is as engineering bacteria pET-31 (b+)-Exendin-4/ BL21 (DE3) plays of reorganization Exendin-4 amalgamation and expression.

The used plasmid of subclone is available from U.S. Novagen company; Bacillus coli DH 5 а and BL21 (DE3) PlyS is respectively available from Beijing Suo Laibao bio tech ltd company and red autumnal leaves bio tech ltd; Used restriction enzyme is available from Dalian precious biological (Takara); Enteropeptidase is available from U.S. Sigma company (Sigma), and plasmid extraction kit adopts the U.S. EZ Bioresearch a small amount of plasmid extraction kit of our company's sale.

Two, the fermentation expression of engineering bacteria

1, seed activation:

(1) seed activation: get frozen engineering bacteria line and containing on the LB agar plate of 100ul/ml penbritin, cultivated 16 hours for 37 ℃.Select well-grown single bacterium colony, be inoculated in the 20ml LB nutrient solution (containing Amp100ul/ml) 37 ℃ of overnight cultures.

(2) seed liquor preparation: get activated seed, be inoculated in the 200ml 2XYT nutrient solution (containing Amp100ul/ml), cultivated 6 hours for 37 ℃ by 5% inoculum size.

(3) 5L ferment tank: seed liquor is inoculated in by 5% inoculum size in the fermentor tank that the MMBL substratum is housed, and 37 ℃, pH is about 7.0 in control, controls dissolved oxygen 30% through ventilation and rotating speed.When OD600 is 20 left and right sides, add IPTG to final concentration be 0.2mM, induce after 10 hours following jar.Centrifugal, collect thalline.

2, culture medium prescription

(1) L BLiquid nutrient medium (one-level is shaken bottle)

(2)MMBL liquid nutrient medium (secondary medium)

3, fermentative prodn step:

(1) choose single bacterium colony to being equipped with in the 30 mL target substratum, 31 ℃, the 200rpm activation is spent the night.30mL bacterium liquid is received in the 200 mL 2XYT substratum, and 32 ℃, 200rpm cultivates about 2.6 hr.

(2) with 10% inoculum size, receive in the 5 L MMBL substratum, 32 ℃, 240rpm cultivates about 3 hr.

(3) receive in the 60 L fermention mediums.

(4) control growing phase pH is 6.8; Make DO >=10% with stirring velocity and air flow; Treat to begin when the concentration of glucose is reduced to 0.2g/L in the substratum feed supplement.Work as OD ₅₅₀Add 13 mL 1M IPTG when reaching 10 left and right sides and induce (final concentration is 0.2mM), slowly transfer to 7.00 to pH, continue to cultivate after 10 hours and finish.

The fermentation record sees the following form

Experimental result:

Growth curve of bacteria figure in Fig. 1 fermenting process.4ml MMBL substratum is poured in the cuvette; Select 550nm wavelength spectrophotometer adjusted zero point for use,, and the different time nutrient solution measured from 0h successively as blank; The big bacteria suspension of concentration is suitably diluted the back with fresh MMBL liquid nutrient medium to be measured; Make its OD value in 0.10.～0.65, the OD value that after dilution, records will multiply by extension rate, is the actual OD value of nutrient solution.

The Fermentation Process of Parameter monitoring result is seen Fig. 2.(1) temperature: be set at 32 ℃; Slight fluctuations (2) rotating speed is arranged in the fermenting process: initial speed setting is 250rpm, and rotating speed and dissolved oxygen interlock are when dissolved oxygen is reduced to the setting Schwellenwert; Rotating speed begins to increase, and maintains finite concentration to guarantee the dissolved oxygen concentration in the fermentor tank.(3) pH: fermentor tank sterilization back pH is 7.15, is adjusted into 6.8 before the inoculation, and IPTG induces during fermentation 5hr, the pH bottom out, control pH between 7.2～7.3 until fermentation ends.(4) dissolved oxygen: fermentation is provided with dissolved oxygen >=10, and with rotating speed and ventilation interlock, when dissolved oxygen was reduced to Schwellenwert, ventilation increased, and rotating speed begins to increase, to keep dissolved oxygen concentration.

The stability analysis result of plasmid sees Fig. 3.To contain the single bacteria culturing cell dilution 10 of plasmid ⁶Doubly, getting 10 milliliters of cultivations, is 10 up to cell concn ⁸CFU/ml, and then with fresh substratum dilution 10 ⁴-10 ⁶Doubly, repeat above operation and accomplish the unsaturation growth of 200 generations up to cell, be in the logarithmic phase growth in order to guarantee cell always, the concentration of cell is come monitoring growth with OD600, and confirms with plate count.In each dilution point, the culturing cell bed board of dilution (contains 5-bromo-4-chloro-3-indolyl β-D-galactoside), with having blue Lac on LBH agar plate ⁺(plasmid-containing) and white white Lac (plasmid-free) come the difference of comparison quantity, the quantity of passage also can be used for identifying the stability of plasmid simultaneously.

The cell intensive amount of plasmid is seen Fig. 4.Content for plasmid in the aspect textual criticism cell from the side; The fermented liquid of each time point is centrifugal; Obtain thalline; Utilize a small amount of plasmid extraction kit to carry out plasmid extraction, the plasmid that extracting obtains detects on 1% Agar glue, and the plasmid that contained capacity in 10 hours in the inner cell is expressed the mRNA of desirable proteins.

The detected result of active cells is seen Fig. 5.The fermented liquid of each time point is centrifugal, obtain thalline, utilize cell counter to detect at microscopically, bed board carries out the flat board cultivation then, and the result that cultivation obtains and the result difference of microscopic examination relatively can obtain the dead cell number.

Three, the purifying of target protein

Utilize continuous flow centrifuge to carry out centrifugation (8000rpm) fermented liquid after the 60L fermentation, obtain the 5000g bacterial sediment, abandon supernatant.The gained thalline cleans 3 times with phosphate buffered saline buffer (pH=7.4), and the thalline after the cleaning carries out fragmentation.

Crushing process 1: ultrasonic disruption:

Power 60% was worked 3 seconds, and 3 seconds at interval, 300 circulations, till ultrasonic liquid became clear, centrifugal 10,000 rpm 10 minutes removed deposition (not broken cell, cell walls etc.), and obtained component carries out check and analysis by SDS-PAGE.

Crushing process 2: cryogenic high pressure is broken

Adopt JN-3000 low-temperature ultrahigh-pressure continuous flow cell crusher; With continuous flow centrifuge and the fragmentation of JN-3000 low-temperature ultrahigh-pressure continuous flow cell with coupling; Can solve microorganism collection, the cell debris after cytoclasis-removal fragmentation, the full-automation of the operating process of collection supernatant (containing soluble protein); Saved manpower greatly, and the time.The key technical indexes: the broken whole process of pressure 200MPa even flow 35ml/ branch (2.1 liters/hour) power supply 220V 1.5KW is carried out in water bath, and is on the rocks.

Fig. 6 is polyacrylamide gel electrophoresis figure.Sampling 20 microlitres add the 2X sample-loading buffer respectively, incubated at room 30 minutes from fermentor tank; Last appearance; In Bole Mini-3 electrophoresis, carry out gel electrophoresis, the result tangible band occurs near being presented at molecular weight 11.92, conforms to its intended purposes molecular weight of albumen.

The processing of nucleic acid impurity:

Add the DNase of 20mg/L, the RNase nucleic acid that is hydrolyzed, hydrolysis temperature is 37 ℃, incubation time is 30min.

Ammonium sulfate precipitation:

Supernatant after then the crusher nucleicacidase being handled carries out fractionation precipitation with gradient ammonium sulfate: the consumption that in constantly stirring, slowly adds required ammonium sulfate; The concentration of ammonium sulfate is respectively (20%; 40%; 60%, 80%), with among the reorganization Exendin-4 (40-80%) of post precipitation dissolving phosphoric acid salt buffer (pH=7.4) again.

Ion exchange chromatography:

Get the cationic exchange coloum QSHP (GE Healthcare) of 5 kilograms of GE Healthcare life science; The dress post; Earlier with 5 column volumes of phosphate buffered saline buffer (pH=7.4) wash-out; Adopt the phosphoric acid buffer (pH=7) of the sodium-chlor that contains different concns (0.1M-0.5M) then, carry out gradient elution (3 column volume/damping fluids.Obtained component carries out check and analysis by SDS-PAGE.

Column chromatography:

According to the molecular weight of Exendin-4 fusion rotein characteristics at～12 KD; The fusion rotein that wash-out is obtained utilizes cut stream to concentrate; Upper prop adopts the GE Healthcare life science molecular sieve Sephadex G-50 medium of 5KG further to separate, and adopts the phosphate buffered saline buffer (50mM of 15L; PH=7) carry out damping fluid, elution volume is 3 column volumes.

Amidation: the enzyme of employing is reorganization glycocoll amidation monooxygenase; Adopt reaction system, 100 mM TES (N-tris [hydroxymethyl] methyl-2-aminoethane sulfonic acid), 4.7 μ M cupric ions; 5.5mM vitamins C damping fluid; 1mU/ml glycocoll amidating enzyme, pH 7.0, and temperature of reaction is 37 ° of C. reaction times: 3 hours.

Affinity chromatography technology one: adopt the IgG-sepharose-fast flow column (GE Healthcare) of GE company, at first use 50 mM Tris of 3 column volumes, pH 7.5; The damping fluid balance pillar of 150 mM NaCI then with the protein upper prop that will dialyse with same buffer, is washed with 1 times of above-mentioned damping fluid of volume and to be used the 5mM sodium acetate then; The unconjugated impurity of damping fluid flush away of pH5.0 is then used the 500mM sodium acetate, and the damping fluid of pH5.0 elutes target protein; Collect, concentrate; With the 20mM Tris-HCl of capacity, 100mM NaCl, pH7.6 dialysis 12 hours.Prepare enzymic hydrolysis.

Enteropeptidase is handled:

The recombination ox intestine kinase light chain subunit of 1 μ l is at 20 ℃, 20mM Tris-HCl, 100mM NaCl, 8h cleavage of fusion proteins fully under the condition of pH7.6.

Affinity chromatography technology two: the reorganization Exendin-4 of above-mentioned processing is combined on the nickel post, and (50mM pH=7), keeps elution fraction, concentrates lyophilize, the Exenatide that obtains recombinating with initial damping fluid flushing dephosphorylation salt buffer earlier.Adopt to increase histone-GB1 fusion rotein wash-out that the method for imidazole concentration (0.1-0.5M) in the damping fluid will be combined on the pillar after above-mentioned the handling and come out, clean pillar and it is regenerated to specifications.

Desalination and enteropeptidase impurity:

Desalination process 1:

According to the characteristics of the proteic molecular weight of Exendin-4 at～4.2 KD; The target components that wash-out is obtained concentrates; Upper prop adopts GE Healthcare life science molecular sieve Sephadex G-25 medium further to separate, and utilizes phosphate buffered saline buffer (50mM; PH=7) carry out damping fluid, desalination.

Desalination process 2:

Take the mode of cut stream ultrafiltration to carry out desalination.The proteic molecular weight of Exendin-4 relatively is fit to ultrafiltration desalination and concentrated at～4.2 KD, and is similar with general ultra-filtration process.The main influence of investigating working pressure, running time and feed liquid flow velocity to ultra-filtration process is for production process provides the technical matters parameter

The Exendin-4 Determination on content:

Adopt BCA determination of protein concentration test kit (BCA Protein Assay Kit) (Thermo Scientific Pierce, the U.S.), operation steps is carried out the mensuration of protein content to specifications.The result shows, the preparation of output 1. BCA working fluids: quantity per sample adds 1 volume BCA reagent B (50:1) by 50 volume BCA reagent A and prepares an amount of BCA

Working fluid, fully mixing.

2. get an amount of BSA protein standard as required, add deionized water and be diluted to 1mg/ml (stoste 5mg/ml), and mixing.

Attention: protein sample is in what solution in principle, and what solution dilution standard substance also should use.But, also can use for for simplicity

Deionized water, 0.9%NaCl or PBS dilution standard article.Protein standard substance after the dilution also can-20 ℃ of prolonged preservation.All give tacit consent in the following steps and adopt deionized water, can select to use diluent according to condition.

3. typical curve is drawn: get 8 cuvettes and be numbered A-H respectively, add reagent by following list data: (this scheme work linearity range is 50-1000ug/ml.）

Final volume 1200ul

4. behind the vibration mixing, placed 30 minutes for 37 ℃.

5. with the light absorption value at spectrophotometric determination 562nm place, be blank with the absorbance value that does not contain BSA.

6. be X-coordinate with protein content (μ g), light absorption value is an ordinate zou, draws typical curve.

7. sample determination: the testing protein sample is diluted to proper concn with deionized water, gets 100 μ l samples, add 1000 μ l BCA

Working fluid was placed 30 minutes for 37 ℃ behind the mixing, was contrast with the A cuvette then, working sample absorption value A562.

8. according to the absorption value that records, on typical curve, can check in the protein content of sample.

The productive rate of Exenatide is about the 500mg/L fermented liquid.

< 110>the Dongguan City wheat bio tech ltd that extends

< 120>a kind of production technique of producing the reorganization Exenatide

<160>?3

<210>?1

<211>?117

<212>?DNA

< 213>artificial sequence

<220>

<222>?（1）…（117）

<400>?1

catggcgaag?gtaccttcac?gagcgatctg?tctaaacaaa?tggaagaaga?agcggttcgt?60?ctgttcatcg?aatggctgaa?gaatggtggt?ccgtccagtg?gtgcgccgcc?gccgtcg?117

<210>?2

<211>?394

<212>?DNA

< 213>artificial sequence

<220>

<222>?（1）…（394）

<400>2

atcggtgatg?tcggcgatat?aggcgccagc?aaccgcacct?gtggcgccgg?tgatgccggc?60

cacgatgcgt?ccggcgtaga?ggatcgagat?cgatctcgat?cccgcgaaat?taatacgact?120

cactataggg?gaattgtgag?cggataacaa?ttcccctcta?gaaataattt?tgtttaactt?180

taagaagga?gatatacatat?gcaccatcat?catcatcatt?cttctggtct?ggtgccacgc?240

ggttctggta?tgaaagaaac?cgctgctgct?aaattcgaac?gccagcacat?ggacagccca?300

gatctgggta?ccgacgacga?cgacaaggcc?atggcgatat?cggatccgaa?ttcgagctcc?360

gtcgacaagc?ttgcggccgc?actcgagcac?caccaccacc?accactgaga?tccg 394

<210>?3

<211>

<212>?DNA

< 213>artificial sequence

<220>

<222>?（1）…（204）

<400>3

atgcaccatc?atcatcatca?catgcagtac?aaggtcatcc?tgaacggcaa?gacgctgaaa 60

ggcgaaacga?cgacggaagc?agtggacgcg?gccaccgcag?aaaaagtggt?taagcagttt?120

ttcaacgata?atggcgtcga?cggtgaatgg?acgtatgatg?acgctaccaa?aacgtttacc?180

gtgacggaag?atgacgatga?caag 204

Claims (4)

1. the production technique of the Exenatide of recombinating comprises the structure of engineering bacteria, the fermentation expression of engineering bacteria and the purifying of target protein, and it is characterized in that: the structure of described engineering bacteria is undertaken by following step:

(1) synthetic of reorganization Exendin-4 polypeptide gene sequence: according to the e. coli codon preferences, the nucleotide sequence of design coding Exendin-4 transform the goal gene shown in SEQ ID No.1 as;

(2) MAG2010 plasmid: get pET31b (+) plasmid; Cut away KSI fusion gene fragment shown in the SEQ ID No.2, be replaced by the gene shown in the SEQ ID No.3, removing restrictions property restriction endonuclease AlwnI restriction enzyme site and methionine(Met) and follow-up sequence; Replacement is with the gene of enteropeptidase restriction enzyme site and target protein; Cut away the histidine mark sequence, add histidine mark, at the two terminators of C-terminal interpolation of target protein in the proteic front of GB1;

(3) structure of engineering bacteria: the gene fragment that obtains according to the order of the two terminator codons of the cracking site-exedin-4-of promotor-6X Histidine-GB1-tag-enteropeptidase; Utilize the CloneEZ test kit to be connected in the MAG2010 plasmid of well cutting; Obtain recombinant plasmid pET-31 (b+)-Exendin-4, with recombinant plasmid pET-31 (b+)-Exendin-4 with CaCl ₂Method transforms and receives thalline BL21 (DE3) plays, in the LB substratum, is cultivating under 37 ℃, collects thalline, and screening positive clone is as engineering bacteria pET-31 (b+)-Exendin-4/ BL21 (DE3) plays of reorganization Exendin-4 amalgamation and expression.

2. the production technique of reorganization Exenatide according to claim 1 is characterized in that: the fermentation expression of described engineering bacteria comprises the steps:

(1) choose single bacterium colony to being equipped with in the 30 mL target substratum, 31 ℃, the 200rpm activation is spent the night;

(2) 30mL bacterium liquid is received in the 200 mL 2XYT substratum, 32 ℃, 200rpm cultivates about 2.6 hr;

(3) with 10% inoculum size, receive in the 5 L MMBL substratum, 32 ℃, 240rpm cultivates about 3 hr;

(4) receive in the 60 L fermention mediums;

(5) control growing phase pH is 6.8; Make DO>=10% with stirring velocity and air flow; Treat to begin when the concentration of glucose is reduced to 0.2g/L in the substratum feed supplement; Work as OD ₅₅₀Add 13 mL 1M IPTG when reaching 10 left and right sides and induce, final concentration is 0.2mM, slowly transfers to 7.00 to pH, continues to cultivate after 10 hours and finishes.

3. the production technique of reorganization Exenatide according to claim 1 and 2 is characterized in that: the purification step of described target protein adopts the affinity chromatography of ion exchange chromatography-column chromatography-sandwich type to separate and purifying; Engineering bacillus by fermentation, the output of the reorganization Exenatide that obtains of purifying reaches the productive rate of 500 mg/litre.

4. the production technique of reorganization Exenatide according to claim 3 is characterized in that: the affinity chromatography of described sandwich type comprises affinity chromatography one, enzymatic lysis and affinity chromatography two, and concrete steps are following:

(1) affinity chromatography one: adopt the IgG-sepharose-fast flow column of GE company, at first use 50 mM Tris of 3 column volumes, pH 7.5; The damping fluid balance pillar of 150 mM NaCI then with the protein upper prop that will dialyse with same buffer, is washed with 1 times of above-mentioned damping fluid of volume and to be used the 5mM sodium acetate then; The unconjugated impurity of damping fluid flush away of pH5.0 is then used the 500mM sodium acetate, and the damping fluid of pH5.0 elutes target protein; Collect, concentrate; With the 20mM Tris-HCl of capacity, 100mM NaCl, pH7.6 dialysis 12 hours;

(2) enteropeptidase is handled: the recombination ox intestine kinase light chain subunit of 1 μ l is at 20 ℃, 20mM Tris-HCl, 100mM NaCl, 8h cleavage of fusion proteins fully under the condition of pH7.6;

(3) affinity chromatography two: the reorganization Exendin-4 of above-mentioned processing is combined on the nickel post, with initial damping fluid flushing dephosphorylation salt buffer, keeps elution fraction earlier, concentrate lyophilize, the Exenatide that obtains recombinating.

Images