In vitro folding, purification and characterization of Escherichia coli outer membrane protease OmpT

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(NO 2 )-NH 2 (where Abz is o-aminobenzoyl and Tyr(NO 2 ) is 3-nitrotyrosine) enabled the determination of the kinetic parameters. The wild-type enzyme has an affinity K m of 0.4 mm for the substrate and a turnover number k cat of 40 s 21. The K m and k cat for the double variant were 1.1 mm and 1.6 s 21 , respectively. The pH profiles of the wild type and variant were identical, showing optimal activity at pH 6.5 and pK a values of 5.6 and 7.5, respectively. Circular dichroism spectra of both enzymes indicated a high content of b-strand conformation, and on that basis a b-barrel topology model is proposed.

Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes

“…7 and Table 2) that emphasize the importance of the water content to the interpretation of the spectra. Besides the dominant b-sheet structure in accordance with the study by Kramer et al (2000) applying CD spectroscopy, a-helical structures were also observed, which seemed to be expressed most strongly at higher water content (Table 1), but are reduced in the presence of lipids. These data seem to be in contradiction to Vandeputte-Rutten et al (2001), who found b-sheet structures for crystallized OmpT 69% , but no a-helical structures (see also Protein Data Bank, no.…”

“…OmpT was recombinantly expressed in E. coli and purified from inclusion bodies as described previously (Kramer et al 2000(Kramer et al , 2002 with some modifications.…”

“…The OmpT proteases, which cleave peptides and proteins preferentially between two consecutive basic amino acid residues (Sugimura and Nishihara 1988;Kramer et al 2000Kramer et al , 2001, have been suggested to be involved, for example, in urinary tract disease (Webb and Lundrigan 1996), since ompT genes were found in clinical isolates of E.coli. Also, it has been proposed that OmpT participates in the degradation of antimicrobial peptides secreted by epithelial cells from the urinary tract (Stumpe et al 1998), which is supported by the observation that the antimicrobial peptide protamine could be degraded by OmpT (Stumpe et al 1998).…”

Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site

“…The internally quenched £uorogenic substrate Abz-Ala-Arg-Arg-Ala-Dap(dnp)-Gly was used in a £uorimetric activity assay, as described previously [4]. Assay conditions were 5 WM substrate, 1 mM Tween 20, 5 mM EDTA, 10 mM Tris, pH 8.3.…”

Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpT

“…The active site is located within a deep groove formed by loops L4 and L5 on the one side and L1, L2, and L3 on the other. The structure also reveals a binding site for a single lipopolysaccharide molecule that appears to be important for the catalytic activity of the enzyme (14,15,16). The finding that peptide hydrolysis is weakly inhibited by certain serine protease inhibitors was the basis for the classification of OmpT and its homologues as a distinct serine protease family.…”