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KMID : 0900920010250040381
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Korean journal of Animal Reproduction
2001 Volume.25 No. 4 p.381 ~ p.388
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Expression and Purification of Mutated Porcine Growth Hormone Binding Protein by Using Site-Directed Mutagenesis in E. coli
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Choi K.-H.
Chung K.-S.
Lee H.-T.
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Abstract
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The present study was designed to obtain porcine growth hormone binding protein (pGHBP) improved biological activation as derived mutation in binding site with growth horlnone (GH). A 756 bp of fragment encoding the extracellular domain of pGHBP gene was cloned from the total RNA of porcine fat tissue by reverse transcriptase polymerase chain reaction (RT-PCR) and created mutation in positions 26 and 122 using site-directed mutagenesis method. Position 26 is one and it is near to get on five potential N-linked glycosylation sites located in the extracellular domain of porcine growth hormone receptor known to have a direct influence on combination with GH. Position 122 is known as one of conformational epitope in bovine. It was over-expressed in E. coli using pET-32(c) expression vector and precisely purified by S-protein agarose and enterokinase. In our results, we was obtained pmGHBP of 30 kDa. It suggests to study the effects of the pmGHBP on cell proliferation in vitro and growth rate in vivo after administration.
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KEYWORD
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GHBP, GH, Site-directed mutagenesis
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