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沼泽红假单胞菌(Rhodopseudomonas palustris)核酮糖-1,5-二磷酸羧化酶/氧化酶基因克隆及其在大肠杆菌中的表达
杜翠红,刘静雯,周集体
作者单位E-mail
杜翠红 集美大学生物工程学院 cuihongdu@jmu.edu.cn 
刘静雯 集美大学生物工程学院  
周集体 大连理工大学环境与生命学院  
摘要:
核酮糖-1,5-二磷酸羧化酶/氧化酶(RubisCO)(EC 4.1.139)是光合细菌通过卡尔文循环固定二氧化碳的关键酶。本文采用PCR 方法, 从沼泽红假单胞菌株No.9 中克隆到RubisCO 基因(cbbM)序列(该序列已提交GenBank, 登录号: GU061327)。采用同源建模法, 建立了该RubisCO 蛋白的三维结构模型, 预测其活性位点。将cbbM 基因亚克隆到表达载体pTV118N 上, 构建表达质粒pTV-CBBM,转化大肠杆菌BL21(DE3), 获得表达菌株BL21(DE3)/pTV-CBBM, 该菌株经IPTG 诱导表达后, 进行SDS-PAGE 检测。采用气相色谱法测定破菌上清中的RubisCO 酶活。结果表明: ①cbbM 基因编码461 个氨基酸, 与沼泽红假单胞菌株DCP3 和DH1的RubisCO蛋白序列相似性分别为98%和99%; ②推测沼泽红假单胞菌No.9 中RubisCO 蛋白的活性中心由Asn112、Lys192、Asp194、Glu195、His288、Arg289、His322、Gly424、Ser369、Gly370 和Gly394 等氨基酸残基组成; ③重组蛋白分子量约为50kDa 左右, 与预测相符; ④破菌上清中的RubisCO 酶比活高于原始菌株中的酶活, 说明目的基因在大肠杆菌中得到了有效表达。
关键词:  沼泽红假单胞菌, 核酮糖-1,5-二磷酸羧化酶/氧化酶(RubisCO), 基因克隆, 大肠杆菌, 重组表达
DOI:10.11693/hyhz201003003003
分类号:
基金项目:福建省科技重点项目资助, 2009N0042 号; 集美大学中青年创新团队专项基金资助, 2006A002 号
CLONING AND EXPRESSION OF THE RHODOPSEUDOMONAS PALUSTRIS RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE GENE IN ESCHERICHIA COLI
DU Cui-Hong,LIU Jing-Wen,ZHOU Ji-Ti
Abstract:
Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RubisCO) (EC 4.1.139) is one of key enzymes of the Calvin cycle. In order to further study the gene structure, enzyme characteristics and site-directed mutagenesis of RubisCO, it is necessary to establish a fast, efficient expression system. In this study, the cbbM gene encoding RubisCO was cloned from Rhodopseudomonas palustris No.9 genomic DNA by PCR. The result of sequencing indicates that the cbbM gene encodes 461 amino acid residues and its homology with those of Rps. palustris DCP3 and DH1 are 98% and 99%, respectively. The cbbM gene has been deposited in the GenBank Data Libraries under the accession number GU061327. The three-dimensional structure of Rps. palustris RubisCO was constructed by homology modeling to predict its active sites. Expression plasmid pTV-CBBM was constructed by inserting the cbbM gene into plasmid pTV118N. The expression plasmid was transformed into Escherichia coli BL21 (DE3) to express recombinant RubisCO (rRubisCO). SDS-PAGE analysis revealed that a protein of about 50kDa was expressed, which was consistent with the predicted molecular mass. The specific activity of rRubisCO was higher than that from wild Rps. palustris, indicating that cbbM gene was efficiently expressed in E. coli.
Key words:  Rhodopseudomonas palustris, RubisCO, Gene clone, Escherichia coli, Recombinant expression
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