产琥珀酸厌氧螺菌磷酸烯醇式丙酮酸羧激酶（pckA）基因的克隆、测序及过表达
Cloning, sequencing, and overexpression of the Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase (pckA) gene.
作者信息
Laivenieks M, Vieille C, Zeikus J G
机构信息
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
出版信息
Appl Environ Microbiol. 1997 Jun;63(6):2273-80. doi: 10.1128/aem.63.6.2273-2280.1997.
Abstract
The phosphoenolpyruvate (PEP) carboxykinase-encoding gene from the anaerobic, CO2-fixing, succinate-producing bacterium Anaerobiospirillum succiniciproducens was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a 532-residue polypeptide with a calculated molecular mass of 58.7 kDa. The sequence of the A. succiniciproducens PEP carboxykinase was similar to those of all known ATP/ADP-dependent PEP carboxykinases. In particular, the A. succiniciproducens enzyme was 67.3% identical and 79.2% similar to the E. coli enzyme. The A. succiniciproducens pckA transcription start site was determined, and putative promoter regions were identified. The recombinant enzyme was overexpressed in E. coli. The purified enzyme was indiscernible from the native enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had the same activity as the native enzyme.
摘要
对厌氧、固定二氧化碳、产琥珀酸的细菌琥珀酸厌氧螺菌中编码磷酸烯醇丙酮酸（PEP）羧激酶的基因进行了克隆、测序，并在大肠杆菌中表达。该基因编码一个由532个氨基酸残基组成的多肽，计算分子量为58.7 kDa。琥珀酸厌氧螺菌PEP羧激酶的序列与所有已知的ATP/ADP依赖性PEP羧激酶的序列相似。特别是，琥珀酸厌氧螺菌的这种酶与大肠杆菌的酶有67.3%的同一性和79.2%的相似性。确定了琥珀酸厌氧螺菌pckA的转录起始位点，并鉴定了推定的启动子区域。重组酶在大肠杆菌中过量表达。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，纯化后的酶与天然酶无法区分，并且具有与天然酶相同的活性。
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