Combinational biosynthesis and characterization of a fluorescent 82β-phycocyanin of Spirulina platensis  

作　　者：GUAN XiangYu ZHANG WeiJie CHI XiaoYuan LIN HanZhi WANG JinFeng QIN Song 

机构地区：[1]School of Ocean Sciences,China University of Geoscience,Beijing 100083,China [2]Key Laboratory of Experimental Marine Biology,Institute of Oceanology,Chinese Academy of Sciences Qingdao 266071,China [3]Graduate University of Chinese Academy of Sciences,Beijing 100049,China [4]Yantai Institute of Coastal Zone Research,Chinese Academy of Sciences,Yantai 264003,China

出　　处：《Chinese Science Bulletin》2012年第25期3294-3299,共6页

基　　金：supported by the Jiangsu Provincial Marine Biotechnology Key Lab Open Foundation (2010HS02);a grant from the National Natural Science Foundation of China (Young Scholars,41006094)

摘　　要：To biosynthesize fluorescent Spirulina platensis (Sp)β -phycocyanin (PC) in Escherichia coli, a BLASTP search for homologs of the cpeS gene, a chromophore lyase, was performed against the Synechocystis sp. PCC 6803 (S6) proteome. A highly homologous gene, slr2049, was obtained from the S6 genome. Sites 82 and 153 in -phycocyanin of Sp were modified by site-directed muta- genesis. Two recombinant expression vectors were constructed and transformed into E. coli BL21: (i) pCDF-cpcB (C153A)- slr2049-sll0583-ho1-pcyA; and (ii) pCDF-cpcB (C82I)-slr2049-sll0583-ho1-pcyA. Lyases encoded by the genes slr2049 and sll0583 catalyzed the linking of Sp 82β -PC to phycocyanobilin (PCB), and fluorescent CpcB (C153A)-PCB was generated. We present a strategy for the co-expression of multiple genes in a single expression vector to identify the function of an unknown gene. Recombinant phycobiliproteins produced on a large scale are promising fluorescent tags for diagnostics and pharmacology.To biosynthesize fluorescent Spirulina platensis （Sp） β-phycocyanin （PC） in Escherichia coli, a BLASTP search for homologs of the cpeS gene, a chromophore lyase, was performed against the Synechocystis sp. PCC 6803 （S6） proteome. A highly homologous gene, sir2049, was obtained from the S6 genome. Sites 82 and 153 in β-phycocyanin of Sp were modified by site-directed mutagenesis. Two recombinant expression vectors were constructed and transformed into E. coli BL21： （i） pCDF-cpcB （C153A）- slr2049-sllO583-hol-pcyA; and （ii） pCDF-cpcB （C821）-slr2049-sllO583-hol-pcyA. Lyases encoded by the genes slr2049 and sl10583 catalyzed the linking of Sp 8213-PC to phycocyanobilin （PCB）, and fluorescent CpcB （C153A）-PCB was generated. We present a strategy for the co-expression of multiple genes in a single expression vector to identify the function of an unknown gene. Recombinant phycobiliproteins produced on a large scale are promising fluorescent tags for diagnostics and pharmacology.

关 键 词：组合生物合成 钝顶螺旋藻 藻蓝蛋白 荧光标记 重组表达载体 表征 大肠杆菌 酶基因 

分 类 号：Q943[生物学—植物学]