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

doi:国家高技术研究发展计划(863计划)资助课题(

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

杨莉莉^1,2,贺巾超¹,董文³,范伟全¹,凡炼炼¹,潘秦¹,牟旭鹏¹,颜炜群¹

1.吉林大学再生医学科学研究所生物化学研究室，吉林长春 130021；2.天津医科大学附属肿瘤医院研究所免疫学研究室，天津300060；3.天津市红桥医院骨科，天津300130

收稿日期:2006-04-12 修回日期:1900-01-01 出版日期:2007-01-28 发布日期:2007-01-28
通讯作者: 颜炜群

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

YANG Li-li¹，2, HE Jin-chao¹, DONG Wen³, FANWei-quan¹,FAN Lian-lian¹,PAN Qin¹,MU Xu-peng¹,YAN Wei-qun¹

1.Department of Biochemistry,Institute of Frontier Medical Sciences, Jilin University, Changchun 130021, China; 2. Deparment of Immunology,Tianjin Cancer Hospital and Institute, Tianjin Medical University ,Tianjin 300060, China;3.Department of Orthopaedics, Tianjin Hongqiao Hospital, Tianjin 300130, China

Received:2006-04-12 Revised:1900-01-01 Online:2007-01-28 Published:2007-01-28
Contact: YAN Wei-qun

摘要/Abstract

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。

关键词: 白蛋白信号肽, 毕赤酵母

Abstract: To explore the method of secretory expression of the natural N-terminal rBPTI with high-level in Pichia pastoris. MethodsHuman 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 identi cal with that of natural material. ConclusionrBPTI 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.

Key words: human serum albumin signal peptide, Pichia pastoris

中图分类号:

Q553

杨莉莉,贺巾超,董文,范伟全,凡炼炼,潘秦,牟旭鹏,颜炜群. 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[J]. J4, 2007, 33(1): 17-20.

YANG Li-li，2, HE Jin-chao, DONG Wen, FANWei-quan,FAN Lian-lian,PAN Qin,MU Xu-peng,YAN Wei-qun. High level secretory expression of natural N-terminal rBPTI with human serum albumin signal peptide in Pichia pastoris[J]. J4, 2007, 33(1): 17-20.

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