点状产气单胞菌脯氨酰内肽酶在大肠杆菌中的高效表达  

High-efficiency Expression of Prolyl Endopeptidase from Aeromonas punctata subsp. punctata in Escherichia coli

作　　者：李民[1] 徐皓[1] 陈常庆[1] 

机构地区：[1]中国科学院上海生物工程研究中心,上海200233

出　　处：《生物化学与生物物理学报》2000年第2期183-186,共4页

基　　金：中国科学院上海分院择优项目!沪院 98 0 2 0 6&&

摘　　要：采用PCR分段克隆法将点状产气单胞菌点状亚种 (Aeromonaspuctatasubsp .puctata)脯氨酰内肽酶 ( pro lylendopeptidase ,apPEP)的编码区基因分成 3段扩增并拼接成编码 690个氨基酸的完整基因apPEP ,将其克隆在表达载体 pBL和 pKKH上 ,构建成温度和IPTG诱导型高效表达apPEP的重组大肠杆菌BL2 1/ pBL PEP和BL2 1/pKKH PEP。诱导后 ,重组apPEP的表达量占菌体总蛋白质的 3 0 %左右 ,活力是野生菌的 10 0倍左右 ,表达产物为可溶性的胞内蛋白 ,非还原SDS PAGE显示为单体 ,分子量为 76kD ,与基因序列预测的分子量一致。采用阴离子HPLC纯化得到了纯度大于 90 %的重组apPEP ,酶比活力为 67u/mg。N端氨基酸序列测定的结果证实了表达的正确性。The open reading frame (ORF) of prolyl endopeptidase gene from Aeromonas punctata subsp. punctata (apPEP) was amplified by PCR in three parts. The amplified DNA segments were ligated to form the complete apPEP gene, and then cloned into expression vectors pBL (temperature inducible) and pKKH (IPTG inducible), respectively. After induction, the expression amounts of recombinant apPEP in BL21/pKKH PEP and BL21/pBL PEP were about 30% of the total bacterial proteins, and the enzyme activities were 100 fold higher than wild strain. Expressed apPEP was mainly soluble intracellular protein. Non reduced SDS PAGE analysis showed that it was a monomer with molecular weight about 76 kD, which corresponded to the prediction from gene sequence. Recombinant apPEP was purified by HPLC, the purity reached 90% and specific activity was 67 u/mg. The N terminal analysis of apPEP demonstrated that the protein sequence was identical as predicted from gene sequence.

关 键 词：脯氨酰内肽酶 大肠杆菌 表达 点状产气单胞菌 

分 类 号：Q55[生物学—生物化学]