Differential secretion and glycosylation of recombinant human chorionic gonadotropin (beta hCG) synthesized using different promoters in the baculovirus expression vector system.
Recombinant baculoviruses vAc beta hCGCOR and vAc beta hCGPOL, carrying the gene (beta hCG) encoding the beta-subunit of human chorionic gonadotropin under the transcriptional control of the late AcNPV core protein gene promoter (PCOR) and the very late polyhedrin gene promoter (PPOL), respectively, were constructed and used to infect lepidopteran cells. Western blot analysis of intra- and extracellular recombinant beta hCG (re-beta hCG) revealed that the secretion of beta hCGCOR was relatively higher. Enzymatic and chemical analysis of carbohydrates showed that beta hCGCOR was more glycosylated than beta hCGPOL. However, the insect-derived beta hCG, with a high-mannose type of sugar, was glycosylated differently and to a lesser extent when compared with the native, urinary beta hCG, and consequently, beta hCGCOR was more bioactive on a unit-mass basis than beta hCGPOL. This temporal gene expression strategy, besides being able to circumvent the 'secretory load' encountered during the synthesis of extensively glycosylated proteins in the baculovirus system, also offers a model to study the role of carbohydrates, both in qualitative and quantitative terms, in protein structure and function.
Sridhar
P
P
National Institute of Immunology, New Delhi, India.
Hasnain
S E
SE
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002241:Carbohydrates / Q000032:analysis; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006031:Glycosylation; D009036:Moths; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D002241:Carbohydrates; D006063:Chorionic Gonadotropin; D011994:Recombinant Proteins