华北农学报Issue(2)：64-71,8.DOI:10.7668/hbnxb.2015.02.013
海带核基因组编码CbbX蛋白的原核表达与纯化
Prokaryotic Expression and Purification of Nulear-genomic cbbX Gene of Saccharina japonica
摘要
Abstract
Calvin-benson-bassham cycle-related enzyme( CbbX)is regarded as red-like RuBisCo activase that activates RuBisCo in Chromophyta. In Saccharina japonica,cbbX gene is encoded in nuclear genome( nuccbbX)and plastid genome(ptcbbX). With RT-PCR,the fragment containing intact ORF of nuccbbX gene and restriction en-zyme cutting site was amplified in this study. Multiple sequence alignment carried out for the protein sequences of nucCbbX,ptCbbX of S. japonica and CbbX of Rhodobacter sphaeroides( RsCbbX)revealed the predicted mature nu-cCbbX contained AAA+ domain,Walker-A and -B motifs and residues related to CbbX functions( ATPase,binding RuBP and keeping hexamer structure stability). The pairwise sequence similarities between nucCbbX and ptCbbX, nucCbbX and RsCbbX were 37. 4% and 36. 8%. While the similarity between ptCbbX and RsCbbX was relatively high 57. 6%. The predicted 3D structure of nucCbbX contained three subdomains which were N-terminal extension consisting of one α-helix,α/β subdomain containing 5 α-helixs and 5 β-foldes,and C-terminal 5-helix bundle sub-domain. For further study the crystal structure of nucCbbX,the target fragment was then successfully subcloned into the express vector pET28a and was expressed in E. coli BL21(DE3)induced with 1% IPTG. The result of SDS-PAGE showed that the fusion protein expressed mainly in the form of inclusion bodies with molecular weight of 50. 2 kDa which is greater than that of nucCbbX mature protein 44. 7 kDa. The fusion proteins were purified after being denatured by urea. And the purified proteins were proven that they could be used in protein crystallization after re-naturation.关键词
海带/核基因组/CbbX/蛋白/表达与纯化Key words
Saccharina japonica/Nuclear genome/CbbX/Protein/Expression and purification分类
生物科学引用本文复制引用
谢玮怡,毕燕会,周志刚..海带核基因组编码CbbX蛋白的原核表达与纯化[J].华北农学报,2015,(2):64-71,8.基金项目
上海海洋大学一流学科海洋科学项目；国家高技术研究发展计划课题项目 （）
