CLONING EXPRESSION AND ACTIVITY ANALYSIS OF A,E,F GENES OF ANABAENA PCC 7120 PEC-OPERON

Phycobiliproteins are brilliantly coloured,intensely fluorescent chromoproteins,which consist of apoproteins and covalently bound linear tetra pyrroles (phycobilins) as prosthetic groups. Phycoerythrocyanin (PEC) is an integral component of the phycobilisome (Pbsome) in several species of cyanobacteria. However,unlike other biliproteins, isolated PEC shows a pronounced reversible photochemistry,which is related to the α-subunit (α-PEC). In this study,the genes pecA, pecE,and pecF from Anabaena PCC 7120 were cloned with vector pBluescript,yielding plasmids pBlu pecA,pBlu pec E and pBlu pec F, respectively. All constructions were verified by sequencing. These genes were subcloned into vector pET30a using the EcoRV and XhoⅠrestriction sites. pBlu PecA, pBlu PecE and pBlu PecF were cleaved with SmalⅠand Xho Ⅰ, and the released genes were ligated to the large pET30a fragment. The E. coli [strain BL21(DE3)]cells containing recombinant pET30a were grown in Luria Bertani medium at 20℃, and harvested 6h after induction with isopropyl thio-β-D galactoside(IPTG). His tagged PecA,E and F were purified separately by metal ion chelating affinity chromatography on chelating sepharose charged with Ni2+. The reconstitution system consists of PCB,His6 PecA,His6 PecE,His6 PecF,and 2 mercaptoethano1. Incubating over night at room temperature,the product of reconstruction investigated by UV vis absorption and fluorescene spectrophotometry appeared an absorption at 565nm,which is the absorption maximum of integral α PEC containing the PVB chromophore. After irradiation at 570 nm,the band at 565 nm decreased,and a new absorption band arose at 505 nm. Some previous experiments showed that the α PEC synthase consists of two proteins,PecE and PecF,in Mastigocladus laminosus. Under catalysis of the over expressed PecE and PecF,PCB can be conVerted to PVB,and bound covalently to apo α PEC to give native α PEC. The classical phototransformation of α PEC involves the reversible shift of the visible absorptiin maximum from 570 to 505nm,which has been attributed to the Z,E isomerization at the 15,16 double bond of phycoviolobilin (PVB) chromophore. The same behavior identifies the PecE and PecF in Anabaena PCC 7120 carry out the same function and the resulting chromoprotein unequivocally is α-PEC.