High expression of the hormone binding active extracellular domain (1-294) of rat lutropin receptor in Escherichia coli

Molecular and Cellular Endocrinology
W Chen, O P Bahl

Abstract

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 rece...Continue Reading

References

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Mar 25, 2010·Proceedings of the National Academy of Sciences of the United States of America·Tyler Paz KormanShiou-Chuan Tsai

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Citations

Mar 1, 1996·Molecular and Cellular Endocrinology·P NarayanD Puett
Jan 1, 1996·Baillière's Clinical Endocrinology and Metabolism·R PaschkeG Vassart
Apr 21, 2010·The Journal of Biological Chemistry·Esther E Biswas-FissSubhasis B Biswas
Apr 29, 1998·Annual Review of Physiology·M L Dufau
Sep 15, 1995·The Journal of Biological Chemistry·R ZhangM L Dufau
Feb 1, 1996·The Journal of Investigative Dermatology·O M MemarB S Prabhakar

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