Expression and Purification of Chitotriosidase in Methylotrophic Yeast Pichia Pastoris

Objective To express and purify human Chitotriosidase in methylotrophic yeast Pichia pastoris and obtain Chitotriosidase protein with natural structure.Methods Gene encoding human Chitotriosidase,amplified by PCR from pUC19-Chit vector,was cloned into the vector pPIC9k.The constructed plasmid was transformed into yeast GS115 by electroporation.The recombinant transformants with a high copy number of the plasmid were selected by using MD plate and G418.The expression of Chitotriosidase was induced by addition of methanol.The supernatant was collected and purified with chitin affinity column chromatography and analyzed by SDS-PAGE.Results The recombinant vector of pPIC9K-Chit was constructed successfully.The SDS-PAGE analysis showed that the Mr of Chitotriosidase was about 50 000.The concentration of the protein was 24 mg/L and the purity was 97%.Conclusion Chitotriosidase gene was expressed successfully in yeast Pichia pastoris.The obtaining of Chitotriosidase protein with natural structure will play an important role in the further study of Chitotriosidase.