人类肿瘤坏死因子相关凋亡配体活性部位基因在毕赤酵母中的表达  

Expression of a DNA Fragment Encoding the Active Domain of Human TNF Related Apoptosis Inducing Ligand in Pichia pastoris

作　　者：徐红[1] 赖心田 叶凯[1] 马辉文[1] 洪葵[3] 

机构地区：[1]武汉大学药学院,武汉430072 [2]海南华康生物制品研究所,海口570310 [3]华南热带农业大学热带作物生物技术国家重点实验室,海口571101

出　　处：《生物工程学报》2003年第2期163-167,共5页Chinese Journal of Biotechnology

摘　　要：探讨了人类肿瘤坏死因子相关凋亡配体 (TRAIL)生物活性部分在巴斯德毕赤酵母 (Pichiapastoris)中的分泌表达。由于TRAIL活性部位区第 149 15 0氨基酸含有一个Kex2位点 ,根据Pichiapastoris分泌表达的特点 ,对 149位氨基酸进行了改造 ,使其编码的氨基酸由Arg突变为Lys。这样使TRAIL在分泌的过程中不被Kex2切割 ,保证了片段的完整。序列分析正确后 ,将编码TRAIL的可溶区的基因片段插入到酵母表达载体pPIC9K中 ,使之位于α 因子信号肽下游 ,且与之同框 ,构建分泌型表达载体pPIC9K TRAIL。采用原生质体法将重组质粒转化Pichiapastoris菌株GS115 ,获基因工程菌株Pichiapastoris(pPIC9K TRAIL)。将工程菌用甲醇诱导培养 3～ 4d ,对摇瓶发酵的培养上清进行SDS PAGE、Westernblot分析和体外生物活性检测 ,结果发现发酵液中分子量约为 19kD和 38kD的蛋白质能被TRAIL多克隆抗体特异性识别 ,且具有在体外诱导肿瘤细胞凋亡的活性。通过薄层扫描分析显示目的蛋白最高可占可溶性总蛋白的 36 %。上述实验结果表明 ,在Pichiapastoris中表达的TRAIL能以寡聚体的形式存在并且具有生物学活性。Human tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) is a member of the tumor necrosis factor(TNF) family of ligands which has been reported in 1995.The TRAIL protein induces apoptosis of certain types of target cells,such as transformed cells that include but are not limited to cancer cells and virus-infected cells but the normal cells.It is a type Ⅱ transmembrane protein and the extracellular domain of TRAIL is the functional domain in induction of cell apoptosis.A gene fragment encoding for the active domain of TRAIL was modified with oligo-nucleotide directed mutagenesis according to the characters of Pichia pastoris expressing vector.Arginine at the position of 149 corresponding to the amino acid residue 531 which might be a potential Kex2 protease processing sites was substituted with Lysine to prevent the expressed protein from the digestion by the protease.After proved with DNA sequencing.the modified gene fragment coding soluble TRAIL domain was inserted into the Pichia pastoris expression vector pPIC9K in the same reading frame with α-factor secreting signal peptide.The recombinant plasmid pPIC9K-TRAIL was transferred into P.pastoris cell by spheroplast transformation.The recombinant yeasts were identified by antibiotic G418 and Southern dot blot.The transformants(His+ Muts)containing multi-copy gene fragment of TRAIL were selected with increasing concentration of G418 and induced with 0.5% methanol in shaking flask to expression the active domain of TRAIL.After inducing for 3～4 days,the proteins in the culture supernatant was assayed with SDS-PAGE and Western blot.Two expressed protein bands whose appearant molecular weight were 19kD and 38kD,respectively,could be specifically recognized by polyclonal antibodies against human TRAIL.The 38kD protein might be a dimers of TRAIL in the culture supernatant.The amount of expressed foreign protein made up to 36% of the total proteins in the culture suprenatant.Biological activity assay,in vitro,indicated that the expressed protein could i

关 键 词：表达 寡核苷酸介导 突变作用 巴斯德毕赤酵母 人类肿瘤坏死因子 凋亡配体 

分 类 号：Q786[生物学—分子生物学] R730[医药卫生—肿瘤]