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Expression of biologically active human pre-procorticotropin releasing hormone in E. coli: characterization and
M G Castro1, B A Spruce, D Savva
1Department of Biochemistry and Physiology, School of Animal and Microbial Sciences, University of Reading, England.
Abstract:
1. Human pre-procorticotropin releasing hormone (CRH) was expressed in E. coli strain TG2 as a fusion protein with beta-galactosidase. 2. A 140 kDa band which corresponded to beta-galactosidase pre-proCRH fusion protein was identified in lysates of TG2 cells harbouring the recombinant plasmid pre-proCRH (10-196) [ph PPC (10-196)] after sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Coomassie Blue staining. The identity of the fusion protein was confirmed by Western blotting and a two-site immunoradiometric assay. 3. Purification of the fusion protein from isolated, washed and solubilized inclusion bodies was achieved by ion-exchange chromatography in the presence of 8 M urea. 4. When comparing the adrenocorticotropin-releasing activity on a molar basis, the potency of the chimeric CRH precursor was 4% of that of synthetic r/h CRH (1-41).