Construction and expression of L-arabinose isomerase (L-AI) in cell- surface of pichia pastoris

Abstract

AraA gene encode L-arabinose isomerase (L-AI). It is an enzyme converting D-galactose to D-tagatose. D-tagatose is is a hexoketose monosaccharide sweetener, which is an isomer of D-galactose and rarely found in nature. It is a potential sweetener which has low calorie. The aim of this study is to construct araA gene in the expression vector pJ912-AGa and expression the protein in the cell-surface of Pichia pastoris GS115. Both vector pJ912-AGa and araA gene was digested with SalI and Kpn2I restriction enzymes then was ligated. The ligation solution had been successfully introduced into Escherichia coli DH5a. Vektor pJ912-AGa-araA was successfully integrated into the genome of P. pastoris GS115. Genetically stable transformed cells have been obtained after selection on zeocin medium up to 1000 μg/mL zeocin. We had successfully syntheized L-AI protein in the P. pastoris GS115. Observation using fluorescence microscopy has proven that successful transformaned cell emit green fluorescence derived from the interaction of functional His6 protein and rabbit polyclonal to 6×His tag® and showed that L-AI protein was expressed successfully in cell-surface of P.pastoris.