title: Improved procedure for a high-yield recovery of enzymatically active recombinant calf chymosin from Escherichia coli inclusion bodies.
abstract: The high-yield recovery of enzymatically active recombinant calf chymosin from Escherichia coli inclusion bodies was achieved by optimization of solubilization and renaturation conditions. The solubilization was carried out in 8 M urea at various pHs, at various temperatures, and for various periods of time. The following values were found optimal: 1 h at 31 degrees C, pH 10.4. For successful correct refolding of solubilized prochymosin molecules it was found to be necessary to dilute the solution into an alkaline buffer (pH 10.7) in such a way that the final concentration of urea did not exceed 0.32 M and that of protein 0.275 mg/ml. Our optimized procedure gives about eight times higher yields of enzymatically active chymosin than the current published methods.
authors.affiliation: Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague.
authors.forename: P J
authors.initials: PJ
authors.lastname: Tichý
authors.forename: F
authors.initials: F
authors.lastname: Kaprálek
authors.forename: P
authors.initials: P
authors.lastname: Jecmen
mesh_terms: D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D012085:Chymosin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D014508:Urea
chemical_list: D011993:Recombinant Fusion Proteins; D014508:Urea; D012085:Chymosin