High expression of the hormone binding active extracellular domain (1-294) of rat lutropin receptor in Escherichia coli.
Using the polymerase chain reaction (PCR) technique, the cDNA fragment corresponding to the receptor coding region for residues 1-294 was prepared from rat lutropin receptor (LHR) cDNA and subsequently subcloned into Escherichia coli expression vector pT7-7. This truncated receptor was efficiently expressed in E. coli as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The recombinant protein present in the inclusion bodies was solubilized in 6 M guanidine-HCl and purified in two successive steps of fast performance liquid chromatography (FPLC) using Superose-12 and Mono Q columns. Refolding of the purified recombinant protein was achieved in 1.5 M guanidine-HCl in the presence of an equimolar proportion of cysteine and cystine. The refolded soluble truncated LHR(1-294) had apparent molecular weights of 33 kDa and 140 kDa under reducing and nonreducing conditions, respectively. The multimeric nature of the extracellular domain of the receptor is believed to be due to its self-association by intermolecular disulfide bond formation since the 1-294 amino acid segment has nine cysteine residues and thus has one or possibly more free sulfhydryl groups. The purified truncated receptor had high binding affinity for human choriogonadotropin (hCG) as indicated by ligand blotting on SDS-PAGE and radioligand receptor assays. The amount of hCG required for 50% inhibition of binding of 125I-hCG to the soluble truncated receptor was 2.7 x 10(-10) M (or 12.25 ng). Similarly, the amount of soluble truncated receptor required for 50% inhibition of 125I-hCG binding to the rat ovarian receptor in the radioligand receptor assay was 58 ng.(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
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Department of Biological Sciences, State University of New York at Buffalo, Amherst, NY 14260.
Bahl
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D006063:Chorionic Gonadotropin / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D007986:Luteinizing Hormone / Q000378:metabolism*; D008969:Molecular Sequence Data; D010053:Ovary / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011869:Radioligand Assay; D051381:Rats; D011974:Receptors, LH / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism
D006063:Chorionic Gonadotropin; D010446:Peptide Fragments; D011974:Receptors, LH; D011994:Recombinant Proteins; D007986:Luteinizing Hormone; D004247:DNA