Purification and crystallization of rhizopuspepsin: The use of nickel chelation chromatography to select for catalytically active species

A new method to obtain pure zymogen-derived peptidases is presented. The ke y strategy is to install a polyhistidine peptide tag on the N-terminus of t he propeptide sequence of a zymogen. After expression, purification, and fo lding of the protein, autocatalytic posttranslational cleavage and filtrati on through a nickel affinity column gives pure, functional peptidase. This method takes advantage of the nickel affinity chromatography system that re moves both zymogen peptide and nonfunctional folded peptidase without the n eed to use external enzymes to remove, often incompletely, the resulting fu sion peptide. This technique was used to prepare the aspartic peptidase rhi zopuspepsin. His-tagged rhizopuspepsinogen was expressed, and the desired p rotein was isolated as inclusion bodies and refolded. The proenzyme was pur ified by normal methods and then the relatively pure proenzyme was activate d via intramolecular proteolysis at low pH, The propeptide and any inactive rhizopuspepsinogen were removed via affinity chromatography. This procedur e yields a highly active rhizopuspepsin in 99% purity, which was demonstrat ed by PAGE, protein sequencing, and X-ray crystallography (1.5 Angstrom) of the isolated peptidase. A new fluorescent assay system is introduced for r hizopuspepsin, utilizing the substrate KPVSY(4-NO3-F)RL. The kinetics const ants were K-m = 3.4 mu M +/- 0.31 and k(cat) = 55 +/- 1.0 s(-1). (C) 1999 A cademic Press.