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Gou X.-H.,Chengdu University of Technology | Liu Y.-Y.,Genekey Biotech. Chengdu Co. | Chen Q.-L.,Chengdu University of Technology | Tang J.-J.,Genekey Biotech. Chengdu Co. | And 5 more authors.
AMB Express | Year: 2012

Bikunin is a proteoglycan exhibiting broad-spectrum inhibitory activity against serine proteases and could potentially suppress tumor cell invasion and metastasis. Here, we have successfully expressed recombinant human bikunin (rh-bikunin) in Pichia pastoris and also established the purification procedure. Different fusion genes of h-UTI and domain I, domain I and domain II, domain I, domain II and domain III of human serum albumin (HSA) were inserted into expression vector pPICZαA. After expressed in shake flask, rh-bikunin was produced in an 30-L fermenter and purified by affinity chromatography and cation exchange chromatography. The final expression levels were 200 mg/L and we got totally 1.08 g (3650 IU/mg) of active purified rh-bikunin (purity is 98%) from 20 L of fermentation broth. The rh-bikunin consists of unique form with molecular masses of 25 kDa, and has the same N-terminals sequence as human native bikunin. This study provided a new method for high level expression of active rh-bikunin by using HSA as fusion parter. © 2012 Springer-Verlag Berlin Heidelberg.

Gou X.,Chengdu University of Technology | Gou X.,Meat Proceeding Key Laboratory of Sichuan Province | Wang W.,Meat Proceeding Key Laboratory of Sichuan Province | Liu D.,Chengdu University of Technology | And 7 more authors.
Chinese Journal of Applied and Environmental Biology | Year: 2010

Trehalose is a multifunctional non-reducing disaccharide and an attractive ingredient in biological product and food. Recently, extensive attention has been made for trehalose production via enzyme transform. In this study, the maltooligosyl trehalose tetrahydrolase (MTHase) gene from Sulfolobus shibatae B12, with genetic codon preference for yeast and lower AT content, was artificially synthesized. The artifical gene was cloned into yeast expression vector pPICZaA, and then overexpressed in Pichia pastoris. The results showed that the yeast strains possessed a highly efficient and stably expression for MTHase production. The enzyme activity level from the secretion recombinant protein reached 800 U/mg protein. The genetic steady efficiency of the engineering P. pastoris strain was 84%. Our results provide a basis for trehalose production via enzyme transform in industrial scale.

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Loading Meat Proceeding Key Laboratory of Sichuan Province collaborators