重组胰岛素样生长因子-1的制备
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摘要
本文建立大肠埃希菌高效表达重组人胰岛素样生长因子-1(recombinant human insulin-like growth factor-1,rhIGF-1)的制备工艺。应用本实验室构建的基因工程菌E.coli BL21(DE3)/pET28a-Sp1-IGF-1,对其进行高密度发酵和IPTG诱导表达,发酵液经双水相萃取获得包涵体,产量为3 g/L;对包涵体进行变性和复性,建立反相HPLC法监测复性过程,复性正确率可达600%;复性液经过柱色谱纯化和冷冻干燥,样品纯度大于98%,总收率为35%。本工艺得到的rhIGF-1,分子质量与理论值一致,其HPLC保留时间、肽图和生物活性均与参比制剂一致。
The aim of this study is to establish a process for production of fusion human insulin-like growth factor-1 in prokaryotic cells.The genetic engineering strain E.coli BL21(DE3)/pET28 a-Spl-IGF-1 constructed in our laboratory was cultured by high density fermentation and induced with IPTG.Then the fermentation broth was dealt with the two-aqueous phase extraction to obtain the inclusion bodies weighting wet 3 g/L.The inclusion bodies were denatured and renatured to form the natural disulfide bond.The HPLC method was developed to monitor the process of renaturation,and the ratio of renaturation in the purified product could reach 60%.Furthermore,the refolding solution was purified via RP-HPLC,and then the product was freeze-dried.The total recovery was 35%.The validity of purified product was verified by mass spectrum,and the biological activity of the product was evaluated by luminescence ATP detection assay.All these results were in accordance with that of the reference drug.
The aim of this study is to establish a process for production of fusion human insulin-like growth factor-1 in prokaryotic cells.The genetic engineering strain E.coli BL21(DE3)/pET28 a-Spl-IGF-1 constructed in our laboratory was cultured by high density fermentation and induced with IPTG.Then the fermentation broth was dealt with the two-aqueous phase extraction to obtain the inclusion bodies weighting wet 3 g/L.The inclusion bodies were denatured and renatured to form the natural disulfide bond.The HPLC method was developed to monitor the process of renaturation,and the ratio of renaturation in the purified product could reach 60%.Furthermore,the refolding solution was purified via RP-HPLC,and then the product was freeze-dried.The total recovery was 35%.The validity of purified product was verified by mass spectrum,and the biological activity of the product was evaluated by luminescence ATP detection assay.All these results were in accordance with that of the reference drug.
引文
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[2]徐伟文,吴岚晓.胰岛素样生长因子1的临床应用[J].国外医学:内分泌学分册,1999,19(6):270.
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[4]吴晓岚,李明.胰岛素样生长因子-1在大肠埃希菌中的表达[J]. Pharm Biotechnol,2001,8(4):185-188.
[5]曹传平,陈晓虹,吴建国,等.胰岛素样生长因子-1基因原核表达载体的构建及表达蛋白的生物学活性[J].中国生物制品学杂志,2008,21(10):828-851.
[6]JAFARI S,BABAEIPOUR V,SEYEDI H A,et al.Recombinant production of mecasermin in E.coli expression system[J].Res Pharm Sci,2014,9(6):453-461.
[7]HART R A,LESTER P M,REIFSNYDER D H,et al.Large scale,in situ isolation of periplasmic IGF-1 from E.coli[J].Biotechnology(NY),1994,10(12):1113-1116.
[8]刘宝英,赵明,王会信,等.人胰岛素样生长因子1在大肠埃希菌中的表达研究[J].中国生物化学与分子生物学,1998,14(1):47-51.
[2]徐伟文,吴岚晓.胰岛素样生长因子1的临床应用[J].国外医学:内分泌学分册,1999,19(6):270.
[3]BUELL G,SCHULZ M F,SEIZER G,et al.Optimizing the expression in E.coli of a synthetic gene encoding somatomedin-C(IGF-1)[J].Nucleic Acids Res, 1985,13(6):1923-1938.
[4]吴晓岚,李明.胰岛素样生长因子-1在大肠埃希菌中的表达[J]. Pharm Biotechnol,2001,8(4):185-188.
[5]曹传平,陈晓虹,吴建国,等.胰岛素样生长因子-1基因原核表达载体的构建及表达蛋白的生物学活性[J].中国生物制品学杂志,2008,21(10):828-851.
[6]JAFARI S,BABAEIPOUR V,SEYEDI H A,et al.Recombinant production of mecasermin in E.coli expression system[J].Res Pharm Sci,2014,9(6):453-461.
[7]HART R A,LESTER P M,REIFSNYDER D H,et al.Large scale,in situ isolation of periplasmic IGF-1 from E.coli[J].Biotechnology(NY),1994,10(12):1113-1116.
[8]刘宝英,赵明,王会信,等.人胰岛素样生长因子1在大肠埃希菌中的表达研究[J].中国生物化学与分子生物学,1998,14(1):47-51.