cDNA cloning of canine common α gene and its co-expression with canine thyrotropin β gene in baculovirus expression system

The common α gene of the canine glycoprotein hormones was cloned, sequenced and co-expressed with the canine thyrotropin β (TSHβ) gene in the baculovirus expression system, and a bioactive recombinant canine TSH was purified. The canine common α gene was cloned from the total RNA extracted from the canine pituitary gland by the reverse transcription polymerase chain reaction (RT-PCR) using primers that were designed based on the consensus sequences from other species. The resulting 476 bp PCR product is consisted of the full coding sequence for the 96 amino acid mature α subunit, and a sequence encoding a 24 amino, acid signal peptide. Homology analysis with other species revealed that the canine common α subunit potentially contains five disulfide bonds and two oligosaccharide chains N-linked to Asn residues located at positions 56 and 82. For expression in the baculovirus expression system, the common α gene was cloned downstream of the p10 promoter of the pAcUW51 transfer vector, and the previously cloned canine TSHβ gene was inserted under the polyhedrin promoter of the same vector. The recombinant virus containing both α and β genes was generated and propagated before being used to transfect the Sf9 insect cells for expression. The medium from the Sf9 cultures, presumably containing canine TSH α and β in native heterodimer confirmation, exhibited TSH bioactivity as indicated in the cAMP stimulation assay in FRTL-5 cells. The expressed recombinant protein was purified from the culture medium with an affinity column that was coupled with IgG purified from the polyclonal antibodies generated against the partially purified native canine TSH. (C) 2000 Elsevier Science Inc.