光学活性藻蓝蛋白β亚基的体内重组  

Recombinant Expression of a Fluorescent βphycocyanin in Escherichia Coli

作　　者：关翔宇[1] 秦松[2] 张伟杰[2] 唐学玺[1] 

机构地区：[1]中国海洋大学海洋生命学院生态学实验室,山东青岛266003 [2]中科院海洋所实验海洋生物学重点实验室,山东青岛266071

出　　处：《中国海洋大学学报（自然科学版）》2008年第4期605-608,578,共5页Periodical of Ocean University of China

基　　金：中国科学院知识创新工程重要方向项目(KZCX2-YW-209;KZCX2-YW-216);国家高新技术研究发展规划项目计划重点项目(2006AA090303);中国科学院创新团队项目资助

摘　　要：在Synechococcussp.PCC 7002中,首先发现了特异性催化脱辅基藻蓝蛋白β亚基(153位)与藻蓝胆素(phyco-cyanobiling,PCB)连接的裂合酶基因cpcT。本实验在Synechocystissp.PCC 6803中找到与cpcT具有高度同源性的基因slr1649。构建2组相容的重组质粒pCDF-cpcB(C82I)或(C153I)-ho1-pcyA和pRSF-slr1649,cpcB(C82I)和cpcB(C153I)分别编码82和153位被突变的脱辅基藻蓝蛋白β亚基,slr1649编码可能的特异性裂合酶,在大肠杆菌体内生成PCB必需的2个基因(ho1编码血红素氧化酶,pcyA编码藻蓝胆素合成酶),将这些基因共同转化到大肠杆菌诱导表达,利用亲和层析柱对可溶性蛋白进行纯化,产物进行SDS-PAGE、色素蛋白锌电泳和光谱检测。结果表明基因slr1649能够特异性催化CpcB(153位)与PCB的连接,产生了具有光学活性的CpcB(C82I)-PCB,为进一步研究螺旋藻藻蓝蛋白的生物活性奠定了基础。CpcT in Synechococcus sp. PCC 7002, characterized as a lyase that attaches PCB to Cys-153 of CpcB was discovered. The highly homologous gene slr1649 was acquired through BLASTP from genome of Synechocystis sp. PCC 6803. Both sites 82 and 153 Cys of apo-β-phycocyanin were mutated by Ile, and the gene slr1649 was eloned to vector pRSF and transferred to E. coli BL21. The entire pathway of a fluorescent β phycocyanin subunit is synthesized from combinational expression of four genes in two compatible pCDF-cpcB （C82I）-hol-pcyA and pRSF-slr1649 in vivo. The recombinant proteins were purified by metal chelating affinity chromatography and then confirmed by SDS-PAGE, absorbance and fluorescence spectrum. This study pro- vides a method for the production of the fluorescent CpcB（C82I）-PCB in biotechnological applications.

关 键 词：C-藻蓝蛋白 定点突变 重组 

分 类 号：TQ936.2[化学工程] Q946[生物学—植物学]