Expression of biologically active human pre-procorticotropin releasing hormone in E. coli: characterization and purification.
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).
Castro
M G
MG
Department of Biochemistry and Physiology, School of Animal and Microbial Sciences, University of Reading, England.
Spruce
B A
BA
Savva
D
D
Lowry
P J
PJ
D003001:Cloning, Molecular; D003346:Corticotropin-Releasing Hormone / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D015151:Immunoblotting; D015592:Immunoradiometric Assay; D011489:Protein Denaturation; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C041645:pro-corticotropin releasing hormone; D003346:Corticotropin-Releasing Hormone; D001616:beta-Galactosidase