cDNA cloning of canine common alpha gene and its co-expression with canine thyrotropin beta gene in baculovirus expression system.
The common alpha gene of the canine glycoprotein hormones was cloned, sequenced and co-expressed with the canine thyrotropin beta (TSH beta) gene in the baculovirus expression system, and a bioactive recombinant canine TSH was purified. The canine common alpha 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 alpha subunit, and a sequence encoding a 24 amino acid signal peptide. Homology analysis with other species revealed that the canine common alpha 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 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 generated and propagated before being used to transfect the Sf9 insect cells for expression. The medium from the Sf9 cultures, presumably containing canine TSH alpha and beta 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.
Yang
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Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA. yang3@niehs.nih.gov
McGraw
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RA
Ferguson
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DC
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000737:chemistry*; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000662:veterinary; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000096:biosynthesis; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004285:Dogs / Q000235:genetics*; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D018274:Electroporation / Q000662:veterinary; D015870:Gene Expression; D015592:Immunoradiometric Assay / Q000662:veterinary; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification; D011863:Radioimmunoassay / Q000662:veterinary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013972:Thyrotropin / Q000737:chemistry / Q000235:genetics*
D017931:DNA Primers; D011994:Recombinant Proteins; D012313:RNA; D013972:Thyrotropin; D004247:DNA; D000242:Cyclic AMP