Meeting Abstract

11.6  Jan. 4  Production and Purification of Recombinant Lamprey Gonadotropin Hormone Beta Chain in Drosophila S2 Cell Line BUSHOLD, G.J.*; FREAMAT, M.; TRANT, J.M.; KAWAUCHI, H.; TAKAHASHI, A.; MORIYAMA, S.; NOZAKI, M.; SOWER, S.A.; Univ. of NH, Durham; Univ. of NH, Durham; UMBI, Baltimore MD; Kitasato Univ, Japan; Kitasato Univ, Japan; Kitasato Univ, Japan; Niigata Univ, Japan; Univ. of NH, Durham gbushold@unh.edu

Lamprey gonadotropin (GTH) has most recently been identified in lampreys (Sower et al. 2006). Low production of glycoprotein hormones in fish species has been problematic in purification of large quantities of active GTH from lamprey tissue. To understand the role of gonadotropin in lampreys, homologous lamprey GTH is being produced by recombinant techniques to be used in future studies. Therefore, our goal is to express lamprey GTH through the use of the Drosophila Expression System (Invitrogen), which has been shown to be capable of producing active recombinant catfish GTH�s. The coding sequence for the lamprey GTHβ chain was cloned into the pGCAP1 plasmid. Primers specific to the 5�and 3�ends were used to amplify the DNA fragment, which was then ligated into the pMT/V5His expression vector. The expression construct was transfected into the S2 cell line using the Calcium Phosphate transfection reagent. In addition, co-transfection of lamprey GTHβ with channel catfish GTHα or human chorionic gonadotropin (hCG) α was also performed. A tethered construct, which linked the lamprey GTHβ to hCGα, tested the importance of interactions between subunits of heterodimers. Preliminary results from lamprey gonadal in-vitro culture showed that concentrated medium containing each of these three transfected complexes elicited a positive increase in estradiol. Due to the success of these initial steps, further purification methods are underway to produce greater yields of lamprey GTH. (Supported by NSF Grant 0421923 to SAS) sasower@unh.edu