Cloning of a novel ovalbumin gene from quail oviduct and its heterologous expression in Pichia pastoris.
An ovalbumin gene was cloned from Chinese quail (Coturnix coturnix) oviduct by RT-PCR and then inserted into the P. pastoris genome under the control of the methanol inducible 5' alcohol oxidase (AOX) promoter. The recombinant P. pastoris strain was demonstrated to be able to efficiently secrete quail ovalbumin by ELISA analysis using a polyclonal antibody raised against quail ovalbumin. The results showed that induction by 0.75% methanol for 48 h led to the synthesis of secreted quail ovalbumin up to a yield of 5.45 g l(-1). The recombinant ovalbumin was further purified into homogeneity through ion exchange and gel filtration chromatography and SDS-PAGE analysis revealed that, compared to natural ovalbumin, the recombinant ovalbumin could have been glycosylated to the similar extent by P. pastoris.
Yang
Shaohui
S
School of Agriculture and Bioengineering, Tianjin University, Tianjin, China.
Chen
Gong
G
Yu
Xinchun
X
Li
Minggang
M
Wang
Jiehua
J
D000818:Animals; D003001:Cloning, Molecular; D003370:Coturnix / Q000235:genetics*; D005260:Female; D006031:Glycosylation; D010047:Ovalbumin / Q000235:genetics* / Q000378:metabolism; D010057:Oviducts / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D010047:Ovalbumin