PMID:10651827

Citation	Kramer, RA, Zandwijken, D, Egmond, MR and Dekker, N (2000) In vitro folding, purification and characterization of Escherichia coli outer membrane protease ompT. Eur. J. Biochem. 267:885-93
Abstract	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.
Links	PubMed
Keywords	Amino Acid Sequence; Base Sequence; Cell Membrane/enzymology; Circular Dichroism; DNA Primers/genetics; Escherichia coli/enzymology; Escherichia coli/genetics; Hydrogen-Ion Concentration; Inclusion Bodies/enzymology; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Folding; Protein Structure, Secondary; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Serine Endopeptidases/chemistry; Serine Endopeptidases/genetics; Serine Endopeptidases/metabolism; Substrate Specificity
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ECOLI:OMPT	involved_in	GO:0006508: proteolysis	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete

References

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