文献类型：期刊文献

中文题名：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

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

作者：秦秀林[1];钱江潮[2];储炬[2]

机构：[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

年份：2013

卷号：29

期号：6

起止页码：140

中文期刊名：生物技术通报

外文期刊名：Biotechnology Bulletin

收录：CSTPCD;;北大核心:【北大核心2011】；CSCD:【CSCD_E2013_2014】；

基金：广西大学科研基金资助项目(XBZ120010)

语种：中文

中文关键词：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

外文关键词：S-adenosylmethionine synthetase Secretory expression Pichia pastoris Spacer peptide Heterologous expression

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。
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.