Construction of a new secretion/expression system of Yarrowia lipolytica and successfully expressing oncogene rho with it

Abstract:

To simplify the construction procedure of Yarrowia lipolytica secretion/expression system, eliminate the antibiotic contamination, and use gene mel (encoding tyrosinase) as a new type reporter for constructing Y. lipolytica secretion/expression vectors. Gene mel was synthesized by assembly PCR, and fused with homologous promoter pTEF, signal sequence XPR2pre and terminator LIP2t by overlap PCR. The resulted fragment was used to construct new type of secretion/expression plasmids of Yarrowia lipolytica expression system, with which human gene rho was expressed. The synthesized mel and pTEF, XPR2pre, LIP2t were successfully fused into gene fragment TXML, which was substituted for the reporter gene ura3d4 of Y. lipolytica plasmids pINA1297 and pINA1297-a, resulting in new type plasmids pINA1297-M and pINA1297-a-M. This new system could screen the yeast recombinant transformants by phenotype, with which the soluble protein Rho was successfully expressed. In this re-search, mel gene was used as a new reporter for the first time in non-conventional yeast expression system, and it also lends a basis for applying mel in other ?eukaryotic expression systems. Additionally, the resulted soluble Rho can be used for further study of its character, structure, functions and the cor-relation between other members of Rho oncogene family.