Construction Of Prokaryotic Expression Vector Of Salvia Miltiorrhiza C4H Gene And Purification Of Recombinant Protein

The water-soluble ingredient metabolism of Salvia miltiorrhiza is mainly carries on through the Rosmarinic acid pathway,which is consist of two parallel branches: phenolypropanoid metabolism pathway and tyrosine metabolism pathway.And in the phenolypropanoid metabolism pathway the phenylalanine ammonialyase (PAL) as well as the cinnamic acid - 4- hydroxylases (C4H) are the response key enzymes.Therefore,the biology research of C4H which is the regulative enzyme of the phenolypropanoid metabolism pathway is able to unravel the phenolic acids biosynthetic pathway of salvia miltiorrhiza,and the accumulation of phenolic acids is come true.In this study,we cloned the CDs of C4H gene of Salvia miltiorrhiza,and the prokaryotic expression vector pET-32a was constructed,then induced C4H gene expression in E.coli BL21(DE3) by IPTG.Then we optimize the induced affected factors,such as tempreture, consistency of IPTG and induced time,getting the best induced condition.Under these conditions we purified the recombinant protein by affinity purification and cutting purification.(1) We designed a pair of oligonucleotide primers and cloned SmC4H gene by PCR technique,get a gene segment about 1 200 bp.The gene sequencing showed,it is the same as what is showed on Genebank.(2) We designed a pair of oligonucleotide primers which is conteined of two restriction enzyme cutting sites,and sub-cloned the SmC4H gene.Then we cut out the gene segment and the vector pET32a with the same restriction endonuclease EcoRⅤand NotⅠ,and ligeted them.Transform the ligation into E.coli DH5αfor double digested identification.The correct plasmid can be transformed into E.coli BL21(DE3) for induced expression. The SDS-PAGE results showed that the recombinant protein mostly expressed as inclusion body,which is about 55 ku.(3) We sreened the factors affected the expression of recombinant protein ,after that ,the protein was massive expression and purified with two pathways. Affinity purification can get high purity ,but lot of loss was existed in binding buffer. We can recover part of it by reunion of binding buffer. Cutting purification with KCl staining is easy and cheap,also get the high purity protein.