| Summary: | 碩士 === 國立臺灣大學 === 漁業科學研究所 === 94 === The aim of this study is to establish Bac-to-Bac Baculovirus protein express system to over-express the recombinant snakehead fish gonadotropins (shfGTHs). We cloned two subunits of shfGTH cDNAs (α and β) into two distinct promoters of pFastBac Dual vector, transformed into DH10 Bac competent cells to produce the Bacmid, and then infected High 5 insect cells to produce the recombinant shfGTHs. The expressed proteins, hopefully folded correctly and glycosylated, will be used for the studies of biochemical characterization, biological activity assay, and crystal structural analysis. Pituitary glands of vertebrates can synthesize and secret two gonadotropins to control the development, growth, and functions of gonad: one is luteinizing hormone (LH), and the other is follicle-stimulating hormone (FSH). Both LH and FSH are composed of two subunits, α- andβ-subunit: α-subunits are common in a species; whileβ-subunits are different and determine the hormonal specificities. The studies of vertebrate FSH and LH showed that twelve cysteines residues are all conserved in their b-subunits to form six disulfide bonds, expect some fish FSHs. Our previous molecular cloning of snakehead fish GTH cDNAs revealed that the third cysteine residues is absent in shfFSH-β; instead, a positionally shifted cysteine is present at the N-terminus, as found in some phylogenetic related fish. The crystal structures of hFSH and hCG showed that the 3rd and 12th cysteines forms an important disulfide bond to wrap theα-subunit, as “seat-belt”. To investigate the role of the 3rd cysteine in shfFSH-β, we expressed the recombinant shfFSH and shfLH by baculovirus system for future crystal structure analysis.
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