In vitro folding, purification and characterization of Escherichia coli outer membrane protease ompT.
OmpT is a protease present in the outer membrane of Escherichia coli. The enzyme was overexpressed without its signal sequence in E. coli using a T7 system, resulting in the accumulation of OmpT as inclusion bodies. After solubilization of the inclusion bodies in urea, the protein could be folded in vitro by dilution in the presence of detergent n-dodecyl-N, N-dimethyl-1-ammonio-3-propanesulphonate. The addition of lipopolysaccharide to the protein was essential to obtain active enzyme. The correctly folded protein was purified to homogeneity by ion exchange chromatography with a 57% overall yield. Autoproteolysis between Lys217-Arg218 was a major problem during purification, but degradation could be abolished by introducing the mutations G216K and K217G. A novel fluorimetric assay using the internally quenched substrate Abz-Ala-Arg-Arg-Ala-Tyr(NO2)-NH2 (where Abz is o-aminobenzoyl and Tyr(NO2) is 3-nitrotyrosine) enabled the determination of the kinetic parameters. The wild-type enzyme has an affinity Km of 0.4 microM for the substrate and a turnover number kcat of 40 s-1. The Km and kcat for the double variant were 1.1 microM and 1.6 s-1, respectively. The pH profiles of the wild type and variant were identical, showing optimal activity at pH 6.5 and pKa values of 5.6 and 7.5, respectively. Circular dichroism spectra of both enzymes indicated a high content of beta-strand conformation, and on that basis a beta-barrel topology model is proposed.
Kramer
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RA
Department of Enzymology and Protein Engineering, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, The Netherlands.
Zandwijken
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D
Egmond
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MR
Dekker
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N
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease