白蛋白信号肽引导天然N-端的rBPTI在毕赤酵母中高效分泌表达  

High level secretory expression of natural N-terminal rBPTI with human serum albumin signal peptide in Pichia pastoris

作　　者：杨莉莉[1] 贺巾超[1] 董文[2] 范伟全[1] 凡炼炼[1] 潘秦[1] 牟旭鹏[1] 颜炜群[1] 

机构地区：[1]吉林大学再生医学科学研究所生物化学研究室 [2]天津市红桥医院骨科,天津300130

出　　处：《吉林大学学报（医学版）》2007年第1期17-20,共4页Journal of Jilin University：Medicine Edition

基　　金：国家高技术研究发展计划(863计划)资助课题(2004AA205020)

摘　　要：目的:探讨在毕赤酵母(Pichia pastoris)中高效分泌表达具有天然N-端序列的rBPTI。方法:将白蛋白信号肽(hsasp)和bpti基因连接并构建真核表达载体pPICZ/hsasp-bpti,电击转化毕赤酵母菌株X-33,用PCR法、SDS-PAGE和胰蛋白酶抑制活性分析筛选阳性转化菌。结果:转染pPICZ/hsasp-bpti的毕赤酵母X-33工程菌能够高效地分泌表达rBPTI;培养上清中rBPTI含量约200 mg.L-1;SDS-PAGE和质谱分析结果显示rBPTI相对分子质量分别为6 500和6 508;rBPTI的N-端测序结果表明其N-端15个氨基酸序列与天然BPTI完全一致;胰蛋白酶活性抑制分析结果表明,抑制常数Ki=(2.6±0.1)×10-9,与天然BPTI一致。结论:hsasp能够在毕赤酵母中高效地引导分泌表达天然N-端氨基酸序列的rBPTI,其表达量达200 mg.L-1。Objective To explore the method of secretory expression of the natural N-terminal rBPTI with highqevel in Pichia pastoris. Methods Human serum albumin signal peptide （hsasp） and bpti genes were ligated and the eukaryon expression plasmid pPICZ/hsasp-bpti was constructed. The recombinant plasmid was transformed into the Pichia pastoris （X-33） via electroporation. The transforming positive strains were screened by PCR, SDS-PAGE and trypsin inhibition experiment. Results The rBPTI was expressed and secreted in X-33. SDS-PAGE and MS showed that the relative molecular mass of rBPTI were respectively 6 500 and 6 508. Sequence analysis of amino acid proved that 15 amino acids at amido-rBPTI were identical with that of natural BPTI. Trypsin inhibition experiment showed that Ki value of rBPTI [ （2.6±0.1）×10^-9 was identical with that of natural material. Conclusion rBPTI with natural N terminal sequence is successfully expressed in Pichia pastoris with hsasp, and the expression level of rBPTI reaches at 200 mg·L^-1.

关 键 词：牛胰蛋白酶抑制剂 白蛋白信号肽 毕赤酵母 

分 类 号：Q553[生物学—生物化学] Q78