SITE-DIRECTED MUTATION OF THE β-SUBUNIT OF PHYCOCYANIN AND PHYCOERYTHROCYANIN AND THE STUDY OF RECONSTITUTION IN VITRO

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 P1,P2 and P3 were used to generate mutant β-PC(C155I),primer P4,P5 and P6 were used to generate β-PEC(C155I). Both of primer P3 and P6 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.