海带核基因组编码CbbX蛋白的原核表达与纯化

doi:10.7668/hbnxb.2015.02.013

摘要/Abstract

摘要： 为研究海带核基因组编码的nucCbbX蛋白的结构和功能,根据海带配子体核基因 cbbX (GenBank登录号:NP_053838)设计含有酶切位点的引物,通过RT-PCR方法获得末端连接Nde Ⅰ、Xho Ⅰ酶切位点的完整ORF。序列保守性分析表明,预测的海带成熟nucCbbX蛋白序列含具有AAA+结构域、Walker-A和Walker-B ATP结合位点及ATP水解酶和RuBisCo活化酶活性相关位点。序列相似度分析表明,海带nucCbbX与其质体基因组编码的ptCbbX和类球红细菌的 Rs CbbX蛋白氨基酸序列相似度均较低,分别为37.4%和36.8%,相比ptCbbX与 Rs CbbX相似度较高为57.6%。以 Rs CbbX蛋白单体晶体结构模型为模板,预测获得的海带nucCbbX蛋白三级结构中N端为一个α/β子域含有5个α螺旋和5个β折叠,C端为α螺旋子域含有5个α螺旋。为进一步研究海带nucCbbX蛋白晶体结构,通过双酶切和连接反应成功构建pET28a- cbbX 原核表达载体,并转化至BL21感受态细胞,获得pET28a- cbbX /BL21重组菌株。利用终浓度为1%的IPTG进行诱导后,重组蛋白主要以包涵体形式存在,分子量为50.2 kDa,较nucCbbX成熟蛋白(44.7 kDa)大。重组蛋白经变性、纯化和复性后用于蛋白结晶,初步获得海带nucCbbX蛋白晶体。

关键词: 海带, 核基因组, CbbX, 蛋白, 表达与纯化

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 (nuc cbbX) and plastid genome (pt cbbX).With RT-PCR, the fragment containing intact ORF of nuc cbbX gene and restriction enzyme 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 (Rs CbbX) revealed the predicted mature nucCbbX 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 Rs CbbX were 37.4% and 36.8%.While the similarity between ptCbbX and Rs CbbX 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 subdomain.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 renaturation.

Key words: Saccharina japonica, Nuclear genome, CbbX, Protein, Expression and purification

中图分类号:

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, 30(2): 64-71. doi: 10.7668/hbnxb.2015.02.013.

XIE Wei-yi, BI Yan-hui, ZHOU Zhi-gang. Prokaryotic Expression and Purification of Nulear-genomic cbbX Gene of Saccharina japonica[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(2): 64-71. doi: 10.7668/hbnxb.2015.02.013.

http://www.hbnxb.net/CN/Y2015/V30/I2/64

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