Expressing of glycolate oxidase gene in pichia pastoris

The fragment of glycolate oxidase gene was cloned into pPIC3.5K (P.pastoris expression vector). The recombinant plasmid was identified by digested with SnaB I and Not I. It was linerized by Sal I and then transferred into the yeast(Pichia pastoris). Positive clones were selected from the medium without histidine. The feature of integration was examined by PCR, using the chromosome genome of the recombinant yeast as a template. The expression of spinach glycolate oxidase gene in Pichia pastoris appeared to be induced by methanol. The molecular weight of expression protein was about 39.8 kD measured by SDS-PAGE and consisted with the date of glycolate oxidase from the literature. Under optimal conditions, the activity of glycolate oxidase enzyme was 40.8 IU/g wet cell and 17 times as high as the ghost. It approved that the recombinant yeast could express spinach glycolate oxidase efficiently.