Expression of recombinant zebrafish follicle-stimulating hormone (FSH) in methylotropic yeast Pichia pastoris.
Pituitary gonadotropin follicle-stimulating hormone (FSH) was identified in fish two decades ago, but its functional importance in fish reproduction remains poorly defined, especially in non-salmonid species. This gap in our knowledge is partially due to the lack of the hormone in pure form in most of the species studied. We describe here the production of two different forms of biologically active recombinant zebrafish FSH (zfFSH and zfFSH(HIS)) using methylotrophic yeast, Pichia pastoris, as the bioreactor. One form (zfFSH) was produced as the molecule closer to the native form, with the two subunits (Cga and Fshb) expressed separately under different promoters. The other form (zfFSH(HIS)) was produced as a single polypeptide, with the cDNAs for the two subunits joined to form a fusion gene that contained a 6X His tag as part of the linker between the two subunits. The culture conditions were optimized for pH and incubation time for maximal production of the proteins. Using a zebrafish FSH receptor (Fshr)-based reporter gene assay, we tested and compared the biological activities of the two forms of recombinant zebrafish FSH. Our results provide useful information for the future production of recombinant gonadotropins in other fish species.
Yu
Xiaobin
X
Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
Lin
Sze-Wah
SW
Kobayashi
Makito
M
Ge
Wei
W
D000818:Animals; D001483:Base Sequence; D019149:Bioreactors / Q000382:microbiology; D019556:COS Cells; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000235:genetics; D005640:Follicle Stimulating Hormone / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014170:Transformation, Genetic; D029961:Zebrafish Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D017931:DNA Primers; D021122:Protein Subunits; D011994:Recombinant Proteins; D029961:Zebrafish Proteins; D005640:Follicle Stimulating Hormone
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