Expression of wild-type and mutant bovine pancreatic ribonuclease A in Escherichia coli.
Open Access
- 15 January 1993
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 90 (2) , 615-619
- https://doi.org/10.1073/pnas.90.2.615
Abstract
Wild-type ribonuclease A and five mutants thereof have been expressed in Escherichia coli as fusion proteins by using a T7 expression system. The five mutants are C[65-72]S, C[40-95]S, C[58-110]S, C[26-84]S, and K41G. The expressed fusion protein formed inclusion bodies which were then cleaved by factor Xa. The cleaved ribonuclease A was isolated as unfolded (sulfonated), soluble protein which was subsequently folded. This expression system can be used to produce mutants of ribonuclease A in yields suitable for folding and structural studies. All four native three-disulfide mutants exhibited enzymatic activity (5-30%), although only two were thermally stable at room temperature, demonstrating that no single native disulfide bond is essential for folding. The K41G mutant was enzymatically inactive with cyclic cytidine monophosphate as substrate.Keywords
This publication has 28 references indexed in Scilit:
- Cis proline mutants of ribonuclease A. I. thermal stabilityProtein Science, 1992
- Use of bacteriophage T7 lysozyme to improve an inducible T7 expression systemJournal of Molecular Biology, 1991
- A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity?Protein Engineering, Design and Selection, 1991
- Influence of an extrinsic crosslink on the folding pathway of ribonuclease A. Conformational and thermodynamic analysis of crosslinked (7-lysine, 41-lysine)-ribonuclease ABiochemistry, 1984
- Generation of β-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coliNature, 1984
- Folding of ribonuclease A from a partially disordered conformation. Kinetic study under folding conditionsBiochemistry, 1982
- Intermediates in the refolding of reduced ribonuclease AJournal of Molecular Biology, 1979
- Kinetics of refolding of reduced ribonucleaseJournal of Molecular Biology, 1977
- Pathways of folding of reduced bovine pancreatic ribonucleaseBiochemistry, 1974
- Some spectrophotometric and polarimetric experiments with ribonucleaseBiochimica et Biophysica Acta, 1957