Expression of human soluble gp190 in yeast Pichia pastoris.
The cDNA of N-terminal extracellular domain of human leukemia inhibitory factor receptor a-subunit (gp190) ,which encoded soluble gp190 (sgp190) ,was cloned into the methylotrophic yeast Pichia pastoris secreted expression vector pPIC9K. The linearized plasmid sgp190-pPIC9K was then integrated with GS115 genome by spheroplasts transformation. MD medium and PCR method were used for screening positive transformants. The transformants (His+ Mut+) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418 and induced by 0.5% methanol in shaking flask to express sgp190. The sgp190 expressed level could reach up to 26% of total proteins in culture supernatant. SDS-PAGE and Western blotting analysis showed that the molecular weight of sgp190 is about 125 kD and could be specifically recognized by polyclonal antibodies against human sgp190,which indicated good antigenicity of this secreted expressed protein. Biological activity assay showed that purified sgp190 could inhibit the effect of LIF on hCG biosynthesis of cytotrophoblast in vitro.
Li
Gang
G
Department of Genetics and Eugenics, Chongqing Medical University, Chongqing 400016, China. lg168sn@sina.com
Wang
Ying-Xiong
YX
Pan
Wei
W
Liu
Xue-Qing
XQ
He
Jun-Lin
JL
Weng
Ya-Guang
YG
Chen
Xue-Mei
XM
D015153:Blotting, Western; D006063:Chorionic Gonadotropin / Q000096:biosynthesis; D005822:Genetic Vectors; D006801:Humans; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D010843:Pichia / Q000235:genetics*; D021122:Protein Subunits; D011506:Proteins / Q000037:antagonists & inhibitors; D018121:Receptors, Cytokine / Q000096:biosynthesis / Q000235:genetics*; D053606:Receptors, OSM-LIF; D011994:Recombinant Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D006063:Chorionic Gonadotropin; D015850:Interleukin-6; C507837:LIF protein, human; C507840:LIFR protein, human; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D021122:Protein Subunits; D011506:Proteins; D018121:Receptors, Cytokine; D053606:Receptors, OSM-LIF; D011994:Recombinant Proteins