Cloning And Expression Of Trehalose Synthase Gene In Pichia Pastoris

Trehalose was widely used in food、medicine and agriculture because of its uniquebiology function in the past few years. Trehalose synthase is kind of intermoleculeglucose transfer enzyme, which could transfer α-1,4glycoside bond of maltose into α-1,1glycoside bond of trehalose. It has a lot of advantages such as short flow path andeasy control in one step produce of trehalose, so trehalose synthase has great applyprospect in industry production of trehalose.This thesis amplifiacted2067bp of trehalose synthase gene by PCR methodwhich specific primers were used, and recombinant technology was used to clone targetgene to expression vector pPICZαA which between EcoRⅠand EcoRⅠrestrictive site,then the recombinant expression plasmid pPICZαA-TreS was constructed. Therecombinant plasmid was transferred into Pichia pastoris GS115and SMD1168, thezeocin was used to select high copy transformants and some colonies were observed atthe concertraion of1000μg/mL. the phenotype of high copy transformants of GS115and SMD1168was identified which proves that all transformants selected are Mut+. Therecombinant was induced by methanol to express recombinant protein in shake,SDS-PAGE and HPLC results showed that a76kDa specific band was detected inintracellular and exhibited the ability to transfer maltose to trehalose, on the other hand,there was nothing found in extracellular.To find out the factors which affect the recombinant protein secrete, we change thecultivate temperature, induce concentration of methanol, pH, total induction time andculture in fermentator to research and the results showed that the recombinant proteincould not secrete in extracellular at the end. The results revealed that there are lots offactors could affect the target protein secretion, due to the secretion mechanism offoreign protein remain not researched clearly, we still need more researches to explainthis unknown problem.