CDNA cloning of canine common alpha gene and its co-expression with caninethyrotropin beta gene in baculovirus expression system

The common alpha gene of the canine glycoprotein hormones was cloned, seque nced and co-expressed with the canine thyrotropin beta (TSH beta) gene in t he baculovirus expression system, and a bioactive recombinant canine TSH wa s purified. The canine common alpha gene was cloned from the total RNA extr acted from the canine pituitary gland by the reverse transcription polymera se chain reaction (RT-PCR) using primers that were designed based on the co nsensus sequences from other species. The resulting 476 bp PCR product is c onsisted of the full coding sequence for the 96 amino acid mature alpha sub unit, and a sequence encoding a 24 amino acid signal peptide. Homology anal ysis with other species revealed that the canine common alpha subunit poten tially contains five disulfide bonds and two oligosaccharide chains N-linke d to Asn residues located at positions 56 and 82. For expression in the bac ulovirus expression system, the common alpha gene was cloned downstream of the p10 promoter of the pAcUW51 transfer vector, and the previously cloned canine TSH beta gene was inserted under the polyhedrin promoter of the same vector. The recombinant virus containing both alpha and beta genes was gen erated and propagated before being used to transfect the Sf9 insect cells f or expression. The medium from the Sf9 cultures, presumably containing cani ne TSH alpha and beta in native heterodimer confirmation, exhibited TSH bio activity as indicated in the cAMP stimulation assay in FRTL-5 cells. The ex pressed recombinant protein was purified from the culture medium with an af finity column that was coupled with IgG purified from the polyclonal antibo dies generated against the partially purified native canine TSH. (C) 2000 E lsevier Science Inc. All rights reserved.