Cloning And Prokaryotic Expression Of The Phenylpropanoid CoA Acyltransferase (BAPT) CDNA Of Taxus Chinensis Var.Mairei

Taxol,a deterpene amide,has been approved for the treatment of cancers by US Food and Drug Administration(FDA) in 1992.It can promote microtubule's aggregation and restrain cell's mitosis. Taxol is widely distributed in the yew trees(Taxus spp.),but yew trees grow slowly and contain only small amount of taxol.It is new way to study taxol biosynthetic pathway, the key enzymes catalyzing this sequence of reactions and the genes encoding these proteins by new means of biochemistry and gene engineering to improve the biology production yields of taxol.According to the reported cDNA sequence of phenylpropanoid CoA acyltransferase (BAPT) in Taxus Cuspidata, a pair of primers were desigened to clone the whole cDNA of BAPT in Taxus chinensis var.mairei by RT-PCR. The full-length cDNA has 1338 bases by sequence analysis. The homology of nucleotides was 97.76% compared to that of Taxus Cuspidata.We subcloned the cDNA into prokaryotic expression vector pET28b(+) with correct reading frame to give pET-BAPT and transformed it into E. coliBL21(DE3).The recombinant BAPT was expressed effectively after induced with IPTG and existed in the form of inclusion body in E.coli. Though we tried to change and optimize the culture conditions of the engineering E.coli ,the soluble BAPT was not obtained in the end.The BAPT was purified from SDS-PAGE gel and was used to immunize rabbit to produce polyclonal antibodies against BAPT. The value of antiserum was 1:105 , which provided useful protein probe for the Western-Blot to determine the BAPT expression in transgenic cell.The BAPT expressed in inlusion body was inactive and had to be renatured in vitro, a series of procedure including isolation-washing- resolution-purification-refolding was done to renaturate it,but we failed after determining the enzyme activity. There are many factors influnencing the renaturation of recombinant protein,and many methods built on the base of many repeats and optimizations are not general.The experiment of BAPT renaturation and activity analysis still need to be further researched.