PROKARYOTIC EXPRESSION AND IDENTIFICATION OF ALLOPHYCOCYANINα SUBUNIT GENE FROM PORPHYRA HAITANENSIS

Allophycocyanins (APC) consist ofαandβsubunits, was one of phycobiliproteins, act as substances of light-havesting and transferring energy to photosystem reaction centers in algal photosynthesis; it also as a kind of bioactive substances applied perspective in quite a lot of fields. Although theapcA andapcB genes encoding allophycocyaninαandβsubunits ofCyanophora paradoxa,Anabaena variabilis, Cyanidium caldarium, Synechocystis6714 and Aglaothamnion neglectum(Rhodophyta) were also cloned, the research related toapc genes in macro-alga has not been reported. In this study, theapcA gene ofPorphyra haitanensis was PCR amplified from pMD-apcAB recombinant plasmid and cloned intoE. coli fusion expression pTO-T7 vector which allows the overexpression of a target protein. The recombinat plasmid pTO-T7-apcA was transformed into E. coli BL21(DE3) and induced in 0.5mmol/ L IPTGin 37℃, the induce product was identified by western blotting and mass spectrum.The results showed that the 486bp sequence ofapcA was successfully inserted into pTO-T7 plasmid. SDS-PAGE analysis of the inclusion ofE. coli carrying pTO-T7-apcA showed a band with molecular mass of 19.7KD in agreement with a fusion APCα protein with the first 12 Nterminaminol amino acids of T7g10, which was identical to what had been anticipated. And the recombinant fusion protein accounted for more than 50% of the totalE. coli protein after 6 hours inducing. The result of western blotting confirmed that the recombinant fusion protein could specifically react with antibody against native APC proteins. And the mass spectrum results proved that the target protein was allophycocyaninαsubunit ofPorphyra haitanensis.