Molecular Cloning Of Pichia Pastoris PMR1 And Construction Of PpPMR1 Null Mutant

As the representative of the second generation of yeast expression system, Pichia Pastoris, a methylotrophic yeast, has become a highly successful and sub-consummate eukaryotic expression system for its high expression, stability and secretion. It possesses many advantages that other expression systems don't have and has been applied to not only industrial manufactures but also foundational researches. But some heterogenous proteins are affected and degraded by endogenous protease in yeast during its secretion expression. Meanwhile the glycosylation levels of human protein or cytokine are still higher.PMR1 (Plasma membrane ATPase related 1) exists in all biosystem, encoding a novel P-type Ca²⁺-ATPase which localized in the Golgi panniculus adiposus, is a important protein for accommodation the concentration of Ca²⁺, and directly influenced the cell growth and the protein secretion. Reports indicated that, several species PMR1 null mutant can remarkable increase the expression level of foreign protein, meanwhile decrease the out-chain glycosylation.Resultingly, our aim is to construct the null mutant to elevate the expresstion level of heterogenous proteins and then reduce the glycosylation level. First, we referred to relevant research approaches and isolated the PMR1 from pichia pastoris via CODEHOP PCR and IPCR methods; and then constructed the PMR1 null mutant; at last using reporter protein, such as HSA, HSA-IFN-α-2b, HSA-GHRH and GA to carry out inducible expression assays respectively.The result shows that the growth of pichia patoris PMR1 null mutant was conspicuously inhibited by EGTA, and the growth defect was overcomed by adding Ca²⁺ and Mn²⁺ into the media. Meanwhile, to compare with pichia patoris GS115, the expression level of the less glycosylation site forigen proteins was reduced and incresed to GS115 expression level. And expression level of the more glycosylation site forigen proteins was increased and was recovered to GS115 expression level when adding the Ca²⁺ into the media. In the low solubility of oxygen, GS115 expression level of the forigen protein was obviously reduced by the decrease of the oxygen solubility, but the PMR1 null mutant was less affected. So we can utilize this characteristic to fermentation at high-cell dendities, in order to improve the GS115...