利用间隔肽促进S-腺苷甲硫氨酸合成酶在毕赤酵母中的分泌表达

生物技术通报 ›› 2013, Vol. 0 ›› Issue (6): 140-146.

利用间隔肽促进S-腺苷甲硫氨酸合成酶在毕赤酵母中的分泌表达

秦秀林¹, 钱江潮², 储炬²

(1.广西大学生命科学与技术学院, 南宁 530004;2.华东理工大学生物反应器工程国家重点实验室, 上海 200237)

收稿日期:2013-06-20 修回日期:2013-06-20 出版日期:2013-06-20 发布日期:2013-06-20
作者简介:秦秀林, 女, 博士, 讲师, 研究方向: 生物化学与分子生物学;E-mail: xiulinqin@gxu.edu.cn
基金资助:
广西大学科研基金资助项目(XBZ120010)

The Influence of Spacer Peptide and C-Terminal His-tag on Activity and Secretion of MAT from Pichia pastoris

Qin Xiulin¹ Qian Jiangchao² Chu Ju²

(1. College of Life Science and Technology, Guangxi University, Nanning 530004;2. State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237)

Received:2013-06-20 Revised:2013-06-20 Published:2013-06-20 Online:2013-06-20

摘要/Abstract

摘要： S-腺苷甲硫氨酸(SAM) 是所有生物体中都存在的一种重要分子, 对维持生物体功能具有重要的作用, 已经被广泛地用于治疗肝脏疾病、关节炎和忧郁症等多种疾病。利用巴斯德毕赤酵母KM71构建基因工程菌K/9KM, 在AOX1启动子调控下分泌表达达观链霉菌(Streptomyces spectabilis) 来源SAM合成酶(MAT) 。为促进MAT的分泌, 在信号肽Kex2p切割位点后引入间隔肽G-Spacer(NTTEEGEPK) , 构建重组菌K/GM, 其重组蛋白(GM) 表达量达到84.7 mg/L, 分泌效率提高了92%, MAT比酶活提高了58%。在重组蛋白GM的C端融合表达His-tag, 构建重组蛋白GMH表达重组菌K/GMH, GMH的MAT比酶活比GM下降13%, 但分泌效率无显著改变。因此, 间隔肽G-Spacer对MAT的酶活及分泌有显著的促进作用, 而His-tag会降低MAT的酶活力但不影响其分泌效率。所构建的MAT分泌表达基因工程菌K/GMH, 在目的蛋白C端融合表达His-tag可减少分离纯化步骤和产物损失, 易于酶促转化法生产SAM。

关键词: S-腺苷甲硫氨酸合成酶, 分泌表达, 毕赤酵母, 间隔肽, 异源表达

Abstract: S-adenosylmethionine synthetase(MAT)catalyzes the synthesis of S-adenosylmethionine(SAM)from L-methionine and ATP in all living organisms. SAM is an important biochemical molecule participating in a variety of biochemical reactions, and has attracted much interest in clinical research because of its potential to cure many diseases, such as depression, liver disease, and osteoarthritis. Accordingly, to develop a simple and effective way to enzymatically synthesize and produce SAM, the MAT gene(sam2)from Streptomyces spectabilis was expressed successfully under the control of the AOX1 promoter in the recombinant yeast Pichia pastoris KM71. Introduction of a spacer peptide(G-Spacer: NTTEEGEPK)after the α-factor, creating a G-spacer of the N-terminal extension of the MAT(GM), greatly increased the MAT spectivity activity of GM to 158% and concomitantly improved the secretion level of the GM by 92%. In contrast, the MAT activity of the recombinant GM with C-terminal His-tag(GMH)was decreased by 13% as compared to that with the GM. Conclusively, the G-Spacer seem to be of crucial importance in determining the secretion efficiency and activity of MAT in P. pastoris. The C-terminal region of the MAT might also be important to the active protein structure.

Key words: S-adenosylmethionine synthetase, Secretory expression, Pichia pastor, is, Spacer peptide, Heterologous expression

秦秀林, 钱江潮, 储炬. 利用间隔肽促进S-腺苷甲硫氨酸合成酶在毕赤酵母中的分泌表达[J]. 生物技术通报, 2013, 0(6): 140-146.

Qin Xiulin, Qian Jiangchao, Chu Ju. The Influence of Spacer Peptide and C-Terminal His-tag on Activity and Secretion of MAT from Pichia pastoris[J]. Biotechnology Bulletin, 2013, 0(6): 140-146.

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