Expression of wild-type and mutant bovine pancreatic ribonuclease A in Escherichia coli.
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.
Laity
J H
JH
Baker Laboratory of Chemistry, Cornell University, Ithaca, NY 14853-1301.
Shimotakahara
S
S
Scheraga
H A
HA
D000595:Amino Acid Sequence; D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D002942:Circular Dichroism; D003594:Cytoplasmic Granules / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000494:pharmacology; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D017510:Protein Folding; D011498:Protein Precursors / Q000096:biosynthesis* / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet
D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D012259:Ribonuclease, Pancreatic; D015951:Factor Xa