CN101586138B - Process for preparing and purifying Exendin-4 from colon bacillus - Google Patents
Process for preparing and purifying Exendin-4 from colon bacillus Download PDFInfo
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- CN101586138B CN101586138B CN2008100498115A CN200810049811A CN101586138B CN 101586138 B CN101586138 B CN 101586138B CN 2008100498115 A CN2008100498115 A CN 2008100498115A CN 200810049811 A CN200810049811 A CN 200810049811A CN 101586138 B CN101586138 B CN 101586138B
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- elutriant
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Abstract
The invention relates to a process for preparing and purifying Exendin-4 from colon bacillus, belonging to the field of genetic engineering. The purified Exendin-4 is obtained by cultivating Exendin-4high-yielding engineering bacterial strain through bacteria, carrying out non-denatured affinity chromatography, and eliminating N-end fusogenic peptide with enterokinase. Compared with the prior art , the invention improves the formulations of lysate and eluent and adopts a gradient elution technique, thereby enhancing the yield and the purity of a target protein; and besides, the invention simplifies the operating procedures, is simple and easy to operate in operating steps and is suitable for large-batch industrial production.
Description
Technical field
The present invention relates to the purifying process of Exendin-4, be specifically related to a kind of technology that from intestinal bacteria, prepares purifying Exendin-4.
Background technology
Utilize gene engineering method preparation to have the successful key of protein of economic worth to be, first screening superior strain, it two is to optimize purification schemes, the purity and the output of raising target protein.Neither of the two can be dispensed.Especially resemble Exendin-4, only the small protein purifying difficulty of 4.2kD is bigger to contain 39 amino-acid residues, molecular weight.Because (7000 yuan/mg of its high economic worths; The special efficacy new drug of 2-type treating diabetes) and in the future be used for suitability for industrialized production; The technology of preparation purifying Exendin-4 is very important from intestinal bacteria, because it has determined the feasibility of output, purity and the suitability for industrialized production of Exendin-4.Qiagene and Invitrogen two big international biotech firms have set up the purifying process of preparation target protein from intestinal bacteria; Comprise microbial culture, cracking, affinitive layer purification target protein and with enteropeptidase excision N end fusogenic peptide etc.; Because it is not enough that this technology also exists on lysate prescription, elutriant prescription and elution process; Therefore output and the purity of target protein, the especially Exendin-4 of this method purifying of usefulness all can not be satisfactory.
Summary of the invention
The objective of the invention is to overcome the deficiency that prior art exists, the simply technology of preparation purifying Exendin-4 from intestinal bacteria of a kind of operating procedure is provided, can obtain the Exendin-4 of high purity, high yield with this technology.
The objective of the invention is to realize through following technical scheme:
(1) construction process of Exendin-4 high-yielding engineering bacterial strain:
(1) obtains Exendin-4 gene shown in SEQ ID No.1 through transforming; Synthetic Exendin-4 gene; Adopt PCR method; With 5 ' GGTACCGACGACGACGACAAGC 3 ' is forward primer; With 5 ' CCATGGTTATCAAGATGGTGGCG 3 ' is reverse primer, adds restriction enzyme Kpn I recognition site at the Exendin-4 gene 5 ' end shown in the SEQ ID No.I; Behind Kpn I recognition site, add the nucleotide sequence GACGACGACGACAAG of coding enteropeptidase recognition site DDDDK, the nucleotide sequence of enteropeptidase recognition site is closely linked to each other with the Exendin-4 gene; Add two continuous terminator codon TGATAA and restriction enzyme Nco I recognition site CCATGG at the Exendin-4 gene 3 shown in the SEQ ID No.I ' end;
(2) structure of cloning vector pMD19-T-Exendin-4: utilize the described improved Exendin-4 gene of pcr amplification step (1), be connected with the pMD19-T carrier behind the purifying, make up cloning vector pMD19-T-Exendin-4;
(3) structure of efficient expression vector pET-32a (+)-Exendin-4:, reclaim the purpose segment with restriction enzyme Kpn I and Nco I double digestion pMD19-T-Exendin-4 plasmid; With restriction enzyme Kpn I and Nco I digestion expression vector plasmid pET-32a (+), reclaim the big fragment of carrier pET-32a (+), the two connection is promptly obtained Exendin-4 expression vector pET-32a (+)-Exendin-4 that recombinates; Expression vector pET-32a (+)-Exendin-4 is transformed among colibacillus engineering BL21 (DE3) pLYS, obtains Exendin-4 high-yielding engineering bacterial strain BL21 (DE3) pLYS-pET-32a (+)-Exendin-4.
(2) technology of preparation purifying Exendin-4 from intestinal bacteria:
(1) mono-clonal is inoculated in 200ml AMP-LB substratum, and 25 ℃ are spent the night to leave standstill and cultivate 12h; At 37 ℃, be cultured to bacterium liquid OD under the 220rpm condition then
600After reaching 0.6, place cooled on ice, add IPTG to concentration be 1mM, under 30 ℃, 220rpm condition, induce 6h, results bacterium liquid is subsequent use in-20 ℃ of preservations.
(2) the bacterium liquid of step (1) being preserved is after melting on ice, and every gram bacterium is precipitated ice bath 30min, ultrasonic disruption with the suspension of 10ml lysate; Consisting of of used lysate: 50mM NaH
2PO4,500mM NaCl, 10mM imidazoles, 0.01%Trition 100,2% glycerine, 10mM beta-mercaptoethanol, 0.1%PMSF, 1mM MgCl
2, 1U RNase, 1U DNase.
(3) with above-mentioned lysate in 4 ℃, the centrifugal 30min of 8000rpm; Supernatant was mixed 4 ℃ of absorption 3h that slowly vibrate by 2: 1 with resin; 4 ℃, the centrifugal 5min of 3000rpm, supernatant discarded.
(4) every 10ml bacterial lysate adds 40ml rinsing damping fluid in above-mentioned resin, 4 ℃ of 30min that slowly vibrate; 4 ℃, the centrifugal 5min of 3000rpm abandons supernatant; With 10ml rinsing damping fluid resuspension resin, transfer in the chromatography column; Collect the last 1.5ml of rinsing damping fluid, be used for identifying whether washing lotion also has foreign protein.The rinsing damping fluid consists of: 50mM NaH2PO4; 500mM NaCl; The 20mM imidazoles; PH 8.0.
(5), collect elutriant I earlier with the flow velocity wash-out of elutriant I with 1.5ml/min; Use the flow velocity wash-out of elutriant II again, collect elutriant II and obtain purified fusion protein with 1.5ml/min; Used elutriant I consists of: 50mM NaH
2PO
4, 500mM NaCl, 40mM imidazoles, 2% glycerine, 10mM beta-mercaptoethanol; Used elutriant II consists of: 50mM NaH
2PO
4, 500mM NaCl, 80mM imidazoles, 2% glycerine, 10mM beta-mercaptoethanol.
(6) every 3mg reorganization Exendin-4 adds the 1U enteropeptidase, 4 ℃ of digestion 6h, with behind enzymic digestion liquid and the mixed with resin absorption 3h, the elutriant that obtains of chromatography column be the Exendin-4 solution of purifying.
In step (5), the volume of used elutriant I is 11ml, and behind the collection 6.5ml, fraction collection 3 is managed every pipe 1.5ml again.
In step (5), the volume of used elutriant II is 9ml, and every pipe 1.5ml collects 6 pipes altogether.
Because the present invention has made improvement to the prescription of lysate and elutriant, adopt gradient elution technology, thereby increased the output and the purity of target protein; The present invention compares with prior art in addition; Simplified schedule of operation, simple operating steps is prone to row, is fit to the industriallization lot production.
Description of drawings
Fig. 1 is the reorganization Exendin-4 that utilizes in the affinity chromatography separation and purification intestinal bacteria, wherein: 1: induce the 6h thalline; 2-8:40mM imidazole concentration elutriant wash-out fusion rotein; 9-14:80mM imidazole concentration elutriant wash-out fusion rotein; 7: purity 74.67%; 8: purity 75.69%; 9: purity 86.39%; 10: purity 98.10%; 11-14: purity 100%.
Fig. 2 is a N end fusogenic peptide of removing reorganization Exendin-4 with enteropeptidase, wherein:
1: reorganization Exendin-4 fusion rotein;
2: fusion rotein digests 6h with enteropeptidase,
3: the Exendin-4 of purifying.
Embodiment
Below in conjunction with embodiment the present invention is elaborated.
1. the preparation of reagent
(1) configuration of lysis buffer 100ml
50mMNaH
2PO
4 0.690g NaH
2PO
4·H
2O(MW?137.99g/mol)
300mM?NaCl 1.754g NaCl(MW?58.44g/mol)
10mM imidazoles 0.068g imidazoles (MW 68.08g/mol)
Transfer pH to 8.0 with NaOH.
Existing with add at present: Trition 100,0.01%; Glycerine, 2%; Beta-mercaptoethanol, 10mM; PMSF, 0.1%; 100mM MgCl
2, 1mM; 100mM MnCl
2, 1mM; RNase, 1U; DNase, 1U
(2) preparation of rinsing damping fluid 100ml
50mM?NaH
2PO
4?0.690g NaH
2PO
4·H
2O(MW?137.99g/mol)
300mM?NaCl 1.754g NaCl(MW?58.44g/mol)
20mM imidazoles 0.068g imidazoles (MW 68.08g/mol)
Transfer pH to 8.0 with NaOH.
Existing with add at present: glycerine, 2%; Beta-mercaptoethanol, 10mM
(3) preparation of elution buffer 100ml
50mM?NaH
2PO
4?0.690g NaH
2PO
4·H
2O(MW?137.99g/mol)
300mM?NaCl 1.754g ?NaCl(MW?58.44g/mol)
250mM imidazoles 0.068g imidazoles (MW 68.08g/mol)
Transfer pH to 8.0 with NaOH.
Existing with add at present: glycerine, 2%; Beta-mercaptoethanol, 10mM
3.Exendin-4 the construction process of high-yielding engineering bacterial strain
(1) transformation of Exendin-4 gene: transform Exendin-4 gene shown in SEQ ID No.1; Synthetic Exendin-4 gene; Adopt PCR method; With 5 ' GGTACCGACGACGACGACAAGC 3 ' is forward primer; With 5 ' CCATGGTTATCAAGATGGTGGCG 3 ' is reverse primer, adds restriction enzyme Kpn I recognition site at the Exendin-4 gene 5 ' end shown in the SEQ ID No.I; Behind Kpn I recognition site, add the nucleotide sequence GACGACGACGACAAG of coding enteropeptidase recognition site DDDDK, the nucleotide sequence of enteropeptidase recognition site is closely linked to each other with the Exendin-4 gene; Add two continuous terminator codon TGATAA and restriction enzyme Nco I recognition site CCATGG at the Exendin-4 gene 3 shown in the SEQ ID No.I ' end;
(2) structure of cloning vector pMD19-T-Exendin-4: utilize the described improved Exendin-4 gene of pcr amplification step (1), be connected with the pMD19-T carrier behind the purifying, make up cloning vector pMD19-T-Exendin-4;
(3) structure of efficient expression vector pET-32a (+)-Exendin-4:, reclaim the purpose segment with restriction enzyme Kpn I and Nco I double digestion pMD19-T-Exendin-4 plasmid; With restriction enzyme Kpn I and Nco I digestion expression vector plasmid pET-32a (+), reclaim the big fragment of carrier pET-32a (+), the two is connected Exendin-4 expression vector pET-32a (+)-Exendin-4 that promptly recombinates; Expression vector pET-32a (+)-Exendin-4 is transformed among colibacillus engineering BL21 (DE3) pLYS, obtains Exendin-4 high-yielding engineering bacterial strain BL21 (DE3) pLYS-pET-32a (+)-Exendin-4.
3. the technology that from intestinal bacteria, prepares purifying Exendin-4
(1) microbial culture: a mono-clonal is inoculated in 200ml AMP-LB substratum, 25 ℃ of static cultivation 12h that spend the night; At 37 ℃, cultivate about 2h, bacterium liquid OD under the 220rpm condition then
600After reaching 0.6, place on ice cooling rapidly, and add IPTG, make its final concentration reach 1mM.Under 30 ℃, 220rpm condition, induce 6h, crude protein output is the highest, and software analysis shows and reaches 15% of bacterioprotein total amount, and is soluble protein.Results bacterium liquid behind 4000rpm, the 4 ℃ of centrifugal 10min, obtains about 1g bacterium, deposition is placed-20 ℃ of preservations, and is subsequent use.
(2) cracking: after melting on ice, every gram bacterium is with the lysate of 4 ℃ of preservations of the 10ml deposition that suspends with the bacterium of step (1)-20 ℃ preservation, and ice bath 30min uses 250W ultrasonic disruption 30min then, (work 5S 5S at intermittence).Consisting of of used lysate: 50mM NaH
2PO4,500mM NaCl, 10mM imidazoles, 0.01% Trition 100,2% glycerine, 10mM beta-mercaptoethanol, 0.1%PMSF, 1mM MgCl
2, 1U RNase, 1U DNase.
(3) absorption: with above-mentioned lysate in 4 ℃, the centrifugal 30min of 8000rpm; Supernatant was mixed 4 ℃ of absorption 3h that slowly vibrate by 2: 1 with resin; 4 ℃, the centrifugal 5min of 3000rpm, supernatant discarded.
(4) foreigh protein removing: every 10ml bacterial lysate supernatant adds 40ml rinsing damping fluid (50mM NaH in resin
2PO
4500mM NaCl; The 20mM imidazoles; PH 8.0), 4 ℃ of 30min that slowly vibrate; 4 ℃, the centrifugal 5min of 3000rpm abandons supernatant; With 10ml rinsing damping fluid resuspension resin, transfer in the chromatography column; Collect rinsing buffer solution elution liquid 1 pipe (1.5ml), be used for identifying whether washing lotion also has foreign protein.The consumption of rinsing damping fluid is 5 times of bacterial lysate.
(5) wash-out: elder generation flows in chromatography column with constant flow pump and adds 11ml elutriant I, and with the flow velocity wash-out of 1.5ml/min, behind the collection 6.5ml, fraction collection 3 is managed every pipe 1.5ml again.The analysis of SDS-PAGE glue and detection of nucleic acids appearance are measured and shown: it is about 75% that last two pipes can obtain purity, the reorganization Exendin-4 fusion rotein (1g bacterium) of the about 1mg/ml of concentration.Used elutriant I consists of: 50mM NaH
2PO
4, 500mM NaCl, 40mM imidazoles, 2% glycerine, 10mM beta-mercaptoethanol.
Flow in chromatography column with constant flow pump again and add the flow velocity wash-out of 9ml elutriant II, part collection, every pipe 1.5ml with 1.5ml/min; Collect 6 pipes altogether, elutriant II wash-out 9ml collects 6 pipes, every pipe 1.5ml; The 1st pipe purity is about 86%, the target protein of the about 1mg/ml of concentration (1g bacterium); Obtain purity more than 98% at the 2-9 pipe, the target protein that the about 1mg/ml-0.2mg/ml of concentration successively decreases successively, this highly purified reorganization Exendin-4 fusion rotein output is the 6.44mg/g bacterium.Used elutriant II consists of: 50mMNaH
2PO
4, 500 mM NaCl, 80mM imidazoles, 2% glycerine, 10mM beta-mercaptoethanol.
(6) remove N end fusogenic peptide with enteropeptidase: in order to remove the fusogenic peptide of target protein N end, at first with the dialyse purified fusion protein 12hr of wash-out of the deionized water of 20 times of volumes.Then, every 3mg reorganization Exendin-4 adds the 1U enteropeptidase, 4 ℃ of digestion 6h, with behind enzymic digestion liquid and the mixed with resin absorption 3h, the elutriant that obtains of chromatography column be the Exendin-4 of purifying.
Plasmid preparation, competent cell preparation, conversion and the gel electrophoresis etc. that relate among the present invention are all carried out according to " molecular cloning " operation steps.
The recovery of digestion with restriction enzyme, DNA and purifying are all operated by the operation instruction of precious biotech firm, and all reagent materials of using are all purchased in commercially available.
< 110>Agricultural University Of He'nan
< 120>technology of preparation purifying Exendin-4 from intestinal bacteria
<160>1
<170>PatentIn?Version?3.3
<210>1
<211>117
<212>DNA
< 213>artificial sequence
<220>
<221>misc_feature
<222>(1)...(117)
< 223>intestinal bacteria preferendum
<400>1
acggtgaag?gtactttcac?ctctgacctg?tctaaacaga?tggaagaaga?agctgttcgt 60
ctgttcatcg?aatggctgaa?aaacggtggt?ccgtcttctg?gtgctccgcc?accatct 117
Claims (1)
1. the technology of a preparation purifying Exendin-4 from intestinal bacteria is characterized in that it is undertaken by following step:
(1) construction process of Exendin-4 high-yielding engineering bacterial strain:
(1) obtains Exendin-4 gene shown in SEQ ID No.1 through transforming; Synthetic Exendin-4 gene; Adopt PCR method; With 5 ' GGTACCGACGACGACGACAAGC 3 ' is forward primer; With 5 ' CCATGGTTATCAAGATGGTGGCG 3 ' is reverse primer, adds restriction enzyme Kpn I recognition site at the Exendin-4 gene 5 ' end shown in the SEQ ID No.I; Behind Kpn I recognition site, add the nucleotide sequence GACGACGACGACAAG of coding enteropeptidase recognition site DDDDK, the nucleotide sequence of enteropeptidase recognition site is closely linked to each other with the Exendin-4 gene; Add two continuous terminator codon TGATAA and restriction enzyme Nco I recognition site CCATGG at the Exendin-4 gene 3 shown in the SEQ ID No.I ' end;
(2) structure of cloning vector pMD19-T-Exendin-4: utilize the described improved Exendin-4 gene of pcr amplification step (1), be connected with the pMD19-T carrier behind the purifying, make up cloning vector pMD19-T-Exendin-4;
(3) structure of efficient expression vector pET-32a (+)-Exendin-4:, reclaim the purpose segment with restriction enzyme Kpn I and Nco I double digestion pMD19-T-Exendin-4 plasmid; With restriction enzyme Kpn I and Nco I digestion expression vector plasmid pET-32a (+), reclaim the big fragment of carrier pET-32a (+), the two connection is promptly obtained Exendin-4 expression vector pET-32a (+)-Exendin-4 that recombinates; Expression vector pET-32a (+)-Exendin-4 is transformed among colibacillus engineering BL21 (DE3) pLYS, obtains Exendin-4 high-yielding engineering bacterial strain BL21 (DE3) pLYS-pET-32a (+)-Exendin-4;
(2) technology of preparation purifying Exendin-4 from intestinal bacteria:
(1) mono-clonal is inoculated in 200ml AMP-LB substratum, and 25 ℃ are spent the night to leave standstill and cultivate 12h; At 37 ℃, be cultured to bacterium liquid OD under the 220rpm condition then
600After reaching 0.6, place cooled on ice, add IPTG to concentration be 1mM, under 30 ℃, 220rpm condition, induce 6h, results bacterium liquid is subsequent use in-20 ℃ of preservations;
(2) the bacterium liquid of step (1) being preserved is after melting on ice, and every gram bacterium is precipitated ice bath 30min, ultrasonic disruption with the suspension of 10ml lysate; Consisting of of used lysate: 50mM NaH
2PO4,500mM NaCl, 10mM imidazoles, 0.01% Trition 100,2% glycerine, 10mM beta-mercaptoethanol, 0.1% PMSF, 1mM MgCl
2, 1U RNase, 1U DNase;
(3) with above-mentioned lysate in 4 ℃, the centrifugal 30min of 8000rpm; Get supernatant and mixed by 2: 1,4 ℃ of absorption 3h that slowly vibrate with the Ni-resin; 4 ℃, the centrifugal 5min of 3000rpm, supernatant discarded;
(4) the rinsing damping fluid of 4 times of volumes of adding bacterial lysate in the resin of step (3), 4 ℃ of 30min that slowly vibrate; 4 ℃, the centrifugal 5min of 3000rpm abandons supernatant; With the rinsing damping fluid resuspension resin of 1 times of bacterial lysate volume, transfer in the chromatography column; Collect the last 1.5ml of rinsing damping fluid, be used for identifying whether washing lotion also has foreign protein; The rinsing damping fluid consists of: 50mM NaH
2PO
4500mMNaCl; The 20mM imidazoles; PH 8.0;
(5), collect elutriant I earlier with the flow velocity wash-out of elutriant I with 1.5ml/min; Use the flow velocity wash-out of elutriant II again, collect elutriant II and obtain purified fusion protein with 1.5ml/min; Used elutriant I consists of: 50mM NaH
2PO
4, 500mM NaCl, 40mM imidazoles, 2% glycerine, 10mM beta-mercaptoethanol; Used elutriant II consists of: 50mM NaH
2PO
4, 500mM NaCl, 80mM imidazoles, 2% glycerine, 10mM beta-mercaptoethanol; The volume of used elutriant I is 11ml, and behind the collection 6.5ml, fraction collection 3 is managed every pipe 1.5ml again; The volume of used elutriant II is 9ml, and every pipe 1.5ml collects 6 pipes altogether;
(6) every 3mg reorganization Exendin-4 adds the 1U enteropeptidase, 4 ℃ of digestion 6h, behind absorption 3h after enzymic digestion liquid and the Ni-mixed with resin, the elutriant that obtains of chromatography column be the Exendin-4 solution of purifying.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101993851A (en) * | 2010-10-21 | 2011-03-30 | 扬子江药业集团北京海燕药业有限公司 | New expression and preparation method of gene-recombinant Exendin-4 |
| CN102618552B (en) * | 2012-04-01 | 2014-08-20 | 东莞市麦亘生物科技有限公司 | Productive technology of recombined exenatide |
| CN105368864B (en) * | 2015-12-24 | 2020-02-21 | 中国科学院海洋研究所 | Method for obtaining soluble lefteye flounder NPY recombinant protein |
| CN109536573A (en) * | 2019-01-24 | 2019-03-29 | 长春万成生物电子工程有限公司 | A kind of Escherichia coli detection kit and its preparation method and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1635117A (en) * | 2004-11-03 | 2005-07-06 | 东莞宝丽健生物工程研究开发有限公司 | Novel process for preparing recombinant Exendin-4 polypeptide |
| CN1668759A (en) * | 2002-05-17 | 2005-09-14 | 协和发酵工业株式会社 | Method of searching substane having antidiabetic activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1668759A (en) * | 2002-05-17 | 2005-09-14 | 协和发酵工业株式会社 | Method of searching substane having antidiabetic activity |
| CN1635117A (en) * | 2004-11-03 | 2005-07-06 | 东莞宝丽健生物工程研究开发有限公司 | Novel process for preparing recombinant Exendin-4 polypeptide |
Non-Patent Citations (2)
| Title |
|---|
| Xiaopu Yin et al..Expression and puriWcation of exendin-4, a GLP-1 receptor agonist,in Escherichia coli..《Protein Expression and Purification》.2005,259-265. * |
| 哈伍德 主编.应用6×His标签和Ni-NTA树脂对重组蛋白进行一步纯化.《DNA及RNA基本实验技术》.2002,354-367. * |
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