Protein Engineering Of Phycobiliprotein β-Subunits Lyase From Anabaena Sp. PCC7120

To study the structure-function relationship of CpeS, by similarity searches and homologous comparison of sequences, the conservative amino acid sites of CpeS were found. Four conservative tryptophan sites were selected to actualize site-directed mutation. Then four mutants, CpeS(W14I), CpeS(W69M), CpeS(W75S) and CpeS (G119C, W140V), were constructed. The lyase activity of these mutants were tested by reconstitution in vivo to study the effects of tryptophan to CpeS. The results shows that: CpeS(W14I) has hardly lyase activity. Comparing with the wild CpeS, the lyase activity of CpeS(W14I), CpeS(W69M), CpeS(W75S) and CpeS(G119C,W140V) is respectively 7%, 47%, 76% and 68%. From this, it's deduced that: the 14th tryptophan residue in CpeS may be essential amino acid to CpeS, and it may locate in the active site of CpeS. Those results provide information to the study on the tryptophan residue function in CpeS. And it's significant to the further study on the structure and function of CpeS. In the Megaprimer PCR operation of molecular cloning, it's found that Taq DNA polymerase has terminal transferase function, which easily causes unwanted mutants. This phenomenon must be considered when we design mutant primers for Megaprimer PCR. And by using the hi-fi DNA polymerase, Pfu DNA polymerase, this problem can be avoided.