BUSHOLD, G.J.*; FREAMAT, M; TRANT, J.M.; KAWAUCHI, H; TAKAHASHI, A; MORIYAMA, S; NOZAKI, M; SOWER, S.A.; University of New Hampshire, Durham; University of New Hampshire, Durham; University of Maryland Biotechnology Institute, Baltimore; Kitasato University, Japan; Kitasato University, Japan; Kitasato University, Japan; Niigata University, Japan; University of New Hampshire, Durham: Production of Recombinant Lamprey Gonadotropin Hormone Beta-Chain in Drosophila S2 Cell Line
Most recently the gonadotropin (GTH) has been identified in lampreys. Due to the low production of glycoprotein hormones in fish species, purification in large quantities has been a major issue. Therefore, our goal is to produce large quantities of lamprey GTH (lGTH) through the use of the Drosophila S2 cell line that has been shown to be capable of producing active recombinant catfish GTH’s. The coding sequence for lGTH β chain, approximately 450 bp in length was cloned into the pGCAP1 plasmid. Primers specific to the 5′ and 3′ ends were used to amplify the DNA fragment, which was then digested and ligated into the expression vector pMT/V5-His. The expression construct was transfected into the S2 cell line by using Effectene Transfection Reagent. In addition, co-transfection of lGTH β with channel catfish GTH (ccGTH) α expression constructs into the S2 cells was also performed. The stable lines expressing ccGTH α and/or lGTH β were selected by addition of blasticidin in the culture medium over a period of 4 weeks. After induction with CuSO4, the culture medium and cells were tested for recombinant protein expression by Western Blot and RT-PCR, respectively. Proteins were concentrated from the medium and purified through IMAC on Ni Sepharose Fast Flow Beads. The recombinant lGTH will be used in future experiments for production of antisera and developement of specific radioimmunoassay for the study of the physiological role of GTH in lamprey. (Supported by NSF Grant 0421923 to S.A. Sower and the University of New Hampshire UROP 2004)
