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

核酮糖-1,5-二磷酸羧化酶/氧化酶(RubisCO)(EC4.1.139)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。

CLONING AND EXPRESSION OF THE RHODOPSEUDOMONAS PALUSTRIS RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE GENE IN ESCHERICHIA COLI

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.