Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris.
Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalently associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hCGalpha and hCGbeta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Co-expression of both subunits in the same cell resulted in secretion of heterodimeric hCG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hCG. The level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermentor. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hCG and is suitable for structure-function studies of the hormone.
Sen Gupta
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Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
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D001483:Base Sequence; D006063:Chorionic Gonadotropin / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D010843:Pichia / Q000235:genetics*; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000235:genetics; D013329:Structure-Activity Relationship
D006063:Chorionic Gonadotropin; D017931:DNA Primers; D021382:Protein Sorting Signals