Denaturation studies on natural and recombinant bovine prochymosin (prorennin)

Abstract

1. Prochymosin in solution in the presence of 8 M-urea is fully unfolded, as indicated by its fluorescence spectrum, fluorescence quenching behaviour and far-u.v. c.d. spectrum. 2. Equilibrium studies on the unfolding of prochymosin and pepsinogen by urea were carried out at pH 7.5 and pH 9.0. The results indicate that the stabilization energies of the two proteins are identical at pH 7.5, but that at pH 9.0 pepsinogen is significantly less stable than prochymosin. 3. Kinetic studies on the unfolding of prochymosin and pepsinogen indicate that the processes can be described by a single first-order rate constant, and that at any given value of denaturant concentration and pH the rate of unfolding of prochymosin is significantly greater than that of pepsinogen. 4. Unfolding of prochymosin by concentrated urea is not fully reversible, unlike that of pepsinogen. Kinetic analysis of the refolding of the proteins suggests the presence of a slow process following unfolding in urea; for pepsinogen this process leads to a slowly refolding form, whereas for prochymosin the slow process in urea leads to a form that cannot refold on dilution of the denaturant. 5. The results provide a rationale for an empirical process for recovery of recombinant prochymosin after solubilization of inclusion bodies in concentrated urea. 6. In all respects studied here, natural and recombinant bovine prochymosin were indistinguishable, indicating that the refolding protocol yields a recombinant product identical with natural prochymosin.

TheYCR079wgene confers a rapamycin-resistant function and encodes the sixth type 2C protein phosphatase inSaccharomyces cerevisiae

“…The inclusion bodies containing His 6 ‐ΔScPTC6p were obtained by centrifugation of the total cell lysate for 10 min at 12 000 g at 4°C. Solubilization of the inclusion bodies was carried out with 8 M urea as previously described (Sugrue et al , 1990). Denatured His 6 ‐ΔScPTC6p proteins in the above solution was refolded and dialyzed as previously described (Rudolph & Lilie, 1996).…”

Engineering the substrate specificity of rhizopuspepsin: The role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1

“…Even though the refolding process of rhizopuspepsin is not fully reversible, denaturation studies with the aspartic proteinase prochymosin have also shown that the recombinant protein is directly comparable to the native enzyme (Sugrue et al, 1990). Because extensive denaturation studies have not been carried out on mutants of the aspartic proteinases, the trends seen in the change in stability of the large library of mutants and their crystal structures may be useful in rationalizing the change in the denaturation parameters seen for the mutants of rhizopuspepsin ( Fig.…”

Multidisciplinary cycles for protein engineering: Site-directed mutagenesis and X-ray structural studies of aspartic proteinases

“…Although these can be easily isolated, subsequent recovery of the entrapped prochymosin requires extensive solubilisation and in vitro refolding, prior to acid activation to produce mature chymosin [51][52][53][54][55]. In the case of chymosin A the additional possibility of autolysis to give the low activity C2 form may lower the yield Eukaryotic expression systems for chymosin include the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis and the filamentous fungus Aspergillus [60,611. In the S.cerevisiae system a high proportion (approximately 80%) of the invertaseprochymosin fusion product was secreted, but the best overall amount of 20 mg 1-1 of activated material was obtained only after the screening of a large number of transformed colonies for super-secreting mutants [62].…”