重组人骨形态发生蛋白4在毕赤酵母中的表达研究Expression of Recombinant Human Bone Morphogenetic Protein 4 in Pichia pastoris
黄义德;林玮;蔡燕惠;张彦定;
摘要(Abstract):
试图建立人骨形态发生蛋白-4在毕赤酵母(Pichia pastoris)中的表达技术.包括:将hBMP4成熟片段从全长cDNA亚克隆到分泌型表达载体质粒pPIC9K多克隆位点上,构建能表达rhBMP4的载体质粒pPIC9K-hBmp4.经转化Pichia pastoris宿主菌株GS115,PCR鉴定基因整合后,摇瓶培养,用甲醇诱导表达.表达上清液经SDS-PAGE和Western-blot分析,结果表明rhBMP4以单体形式分泌到发酵上清液中,单体分子质量大小约为26 ku,摇瓶表达量达到20.13μg/mL.
关键词(KeyWords): 人骨形态发生蛋白-4;毕赤酵母;重组表达
基金项目(Foundation): 福建省教育厅重点资助项目(JA01015);; 福建省发改委基金资助项目(省发改[2004686号]);; 福建省自然科学基金资助项目(C0410008)
作者(Authors): 黄义德;林玮;蔡燕惠;张彦定;
参考文献(References):
- [1]Sellers R S,Peluso D,Morris E A.The effect of recombinant human bone morphogenetic protein-2(rhBMP-2)onthe healing of full-thickness defects of articular cartilage[J].J Bone Joint Surg Am,1996,10:1452-1463.
- [2]Riley E H,Lane J M,Urist M R,et al.Bone morphogenetic protein-2:biology and applications[J].Clin orthop,1996,324:39-46.
- [3]Shimasaki S,Moore R K,Otsuka F,et al.The bone morphogenetic protein system in mammalian reproduction[J].Endocrine Reviews,2004,25(1):72-101.
- [4]熊绍虎,余磊,潭海燕,等.重组人骨形态发生蛋白-2在大肠杆菌中的表达及纯化[J].第一军医大学学报,2002,22(5):413-416.
- [5]Sampath T K,Maliakal J C,Hauschka P V,et al.Recombinant human osteogenic protein-1 induce new boneformation in vivo with a special activity comparable with natural bovine osteogenic protein and stimulates osteoblastproliferation and differentiation in vitro[J].J Biol Chem,1992,267(28):20352-20362.
- [6]Israel D I,Nove J,Kerns K M,et al.Heterodimeric bone morphogenetic proteins show enhanced activity in vitroand in vivo[J].Growth Factor,1996,13:291-300.
- [7]Cereghino J L,cregg J M.Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J].FEMSMicrobiology Reviews,2000,24:45-66.
- [8]Macauley-Patrick S,Fazenda M L,McNeil B,et al.Heterologous protein production using the Pichia pastorisexpression system[J].Yeast,2005,22:249-270.
- [9]Choi B K,Bobrowicz P,Davidson R C,et al.Use of combinatorial genetic libraries to humanize N-linkedglycosylation in the yeast Pichia pastoris[J].PNAS,2003,100:5022-5027.
- [10]Hamilton S R,Bobrowicz P,Bobrowicz B,et al.Production of complex human glycoprotein in yeast[J].Science,2003,301:1244-1246.
- [11]Romanos M.Advances in the use of Pichia pastoris for high level gene expression[J].Curr Opin Biotechnol,1995,6:527-533.
- [12]Cereghino G L,Cereghino J L,Iigen C,et al.Production of recombinant proteins in fermenter cultures of theyeast Pichia pastoris[J].Current Opinion in Biotechnology,2002,13:329-332.
- [13]Sharp P M,Tuohy T M,Mosurski K R.Codon usage in yeast:cluster analysis clearly differentiates highly andslowly expressed genes[J].Nucleic Acids Research,1986,14(13):5123-5143.
- [14]赵翔,霍克克,李育阳.毕赤酵母的密码子用法分析[J].生物工程学报,2000,16(3):308~311.
- [15]陈凌,黄义德,张彦定.人骨形态发生蛋白7(hBMP7)在毕赤酵母中的分泌表达[J].生物工程学报,2006,22(6):907-913.