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 key strategy is to install a polyhistidine peptide tag on the N-terminus of the propeptide sequence of a zymogen. After expression, purification, and folding of the protein, autocatalytic posttranslational cleavage and filtration through a nickel affinity column gives pure, functional peptidase. This method takes advantage of the nickel affinity chromatography system that removes both zymogen peptide and nonfunctional folded peptidase without the need to use external enzymes to remove, often incompletely, the resulting fusion peptide. This technique was used to prepare the aspartic peptidase rhizopuspepsin. His-tagged rhizopuspepsinogen was expressed, and the desired protein was isolated as inclusion bodies and refolded. The proenzyme was purified by normal methods and then the relatively pure proenzyme was activated via intramolecular proteolysis at low pH. The propeptide and any inactive rhizopuspepsinogen were removed via affinity chromatography. This procedure yields a highly active rhizopuspepsin in 99% purity, which was demonstrated by PAGE, protein sequencing, and X-ray crystallography (1.5 A) of the isolated peptidase. A new fluorescent assay system is introduced for rhizopuspepsin, utilizing the substrate KPVSY(4-NO(3)-F)RL. The kinetics constants were K(m) = 3.4 microM +/- 0.31 and k(cat) = 55 +/- 1.0 s(-1).
Department of Chemistry and School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
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Flentke
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Glinski
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Satyshur
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D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D002614:Chelating Agents / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D003460:Crystallization; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D002614:Chelating Agents; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D009532:Nickel; D016282:Aspartic Acid Endopeptidases; C035794:rhizopuspepsin