摘要
为研究藻蓝蛋白 (PC)和藻红蓝蛋白 (PEC) β亚基 (分别简称为 β PC、β PEC)生物合成、结构与功能的关系 ,用MegaprimerPCR定点突变技术设计 β PC、β PEC中与藻蓝胆素连接的第二个半胱氨酸的定点突变蛋白质 β PC(C15 5I)和 β PEC(C15 5I)。将相应的基因片段亚克隆于表达载体pET 30a,并转化大肠杆菌BL2l(DE3)。经IPTG诱导后 ,β PC(C15 5I)和 β PEC(C15 5I)在大肠杆菌中均得到了高效表达。β PC(C15 5I)和 β PEC(C15 5I)与藻蓝胆素的重组结果表明两个突变体的结构基本没发生改变 ,有利于对 β PC和 β PEC的生物合成进行进一步研究。
To study the structure-function relationships of the β subunit of phycocyanin(β-PC)and phycoerythrocyanin(β-PEC),site-directed mutation was used to generate two mutants,β-PC(C155I)and β-PEC(C155I). The six primers were designed,of which primer P 1,P 2 and P 3 were used to generate mutant β-PC(C155I),primer P 4,P 5 and P 6 were used to generate β-PEC(C155I). Both of primer P 3 and P 6 comained a restriction site EcoRⅤ, so we can pick out the cysteine mutants from the reconstructed plasmids.The PCR products were purified and digested with Sma Ⅰ and XhoⅠ, then recombined with pBluescript vector and transfonned into the E.coli TG1 strain. It was confirmed that the genes were inserted into pBluescript vector by the restriction enzyme double digestion with Sma Ⅰ and Xho Ⅰ,Sma Ⅰ and EcoRⅤ,PCR and sequencing.The genes of the two cysteine mutants were purified after the Sma Ⅰ/Xho Ⅰ double digestion of the reconstructed plasmids,and then inserted into expression vector pET 30a,finally transformed into E. coli BL21(DE3).In the transformed BL21(DE3)strains,24kD protein were overexpressed,respectively.The overexpressed proteins were direct used to reconstitution with pigment PCB in vitro.The purified reconstituted products were determined with absorption spectra and fluorescence spectra,showing that they were similar to the β-PC and β-PEC in spectrum characteristics. It was analysed by GorⅣtoo, showing that there was no other change happened in the secondary structure of β-PC and its mutant,except that Cys-155 of β-PC was situated at the C-terminal of random coil of ANDPNGITKGDC amino acid residues,whereas at N-terminal of alpha helix of ISALISEVASYFDRA in β-PC(C155I).And the similar situation happened to β-PEC and its mutant.So the structure of the two mutants was similar to the wild-type proteins.
出处
《水生生物学报》
CAS
CSCD
北大核心
2004年第4期344-348,共5页
Acta Hydrobiologica Sinica
基金
国家自然科学基金资助项目 ( 3 0 2 70 3 2 6和 90 2 0 10 0 1)
