The plasmid F OmpP protease, a homologue of OmpT, as a potential obstacle to <i>E. coli</i>‐based protein production

Matsuo, Eiichi; Sampei, G.; Mizobuchi, Kiyoshi; Ito, Koreaki

doi:10.1016/s0014-5793(99)01418-0

Cited by 20 publications

(15 citation statements)

References 26 publications

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“…The ompP gene of FЈ plasmid in E. coli XL1-Blue was cloned into pUC19 under the control of its native promoter (18), resulting in plasmid pOP19. E. coli BL21(DE3), a strain that lacks both the ompP and ompT genes, was used for OmpP expression.…”

Section: Purification Of Omppmentioning

confidence: 99%

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Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpP

Hwang

Varadarajan

et al. 2007

J Bacteriol

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Escherichia coliOmpP is an F episome-encoded outer membrane protease that exhibits 71% amino acid sequence identity with OmpT. These two enzymes cleave substrate polypeptides primarily between pairs of basic amino acids. We found that, like OmpT, purified OmpP is active only in the presence of lipopolysaccharide. With optimal peptide substrates, OmpP exhibits high catalytic efficiency (k cat /K m ‫؍‬ 3.0 ؋ 10 6 M ؊1 s ؊1 ). Analysis of the extended amino acid specificity of OmpP by substrate phage revealed that both Arg and Lys are strongly preferred at the P1 and P1 sites of the enzyme. In addition, Thr, Arg, or Ala is preferred at P2; Leu, Ala, or Glu is preferred at P4; and Arg is preferred at P3. Notable differences in OmpP and OmpT specificities include the greater ability of OmpP to accept Lys at the P1 or P1, site as well as the prominence of Ser at P3 in OmpP substrates. Likewise, the OmpP P1 site could better accommodate Ser; as a result, OmpP was able to cleave a peptide substrate between Ser-Arg about 120 times more efficiently than was OmpT. Interestingly, OmpP and OmpT cleave peptides with three consecutive Arg residues at different sites, a difference in specificity that might be important in the inactivation of cationic antimicrobial peptides. Accordingly, we show that the presence of an F episome results in increased resistance to the antimicrobial peptide protamine both in ompT mutants and in wild-type E. coli cells.

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Section: Purification Of Omppmentioning

confidence: 99%

“…The OmpP gene of F plasmids (18) encodes an outer membrane protease consisting of 292 amino acid residues. OmpP belongs to the omptin family of proteases, which includes the Escherichia coli OmpT, Shigella flexneri SopA, Yersinia pestis Pla, Salmonella enterica PgtE, and Erwinia pyrifoliae PlaA enzymes (13).…”

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Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpP

Hwang

Varadarajan

et al. 2007

J Bacteriol

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“…Revised PgtE contains a C-terminal phenylalanine residue that is essential for outer membrane insertion of trimeric OMPs (46). E. coli contains two highly homologous genes encoding OmpT-like proteases: ompT on the chromosome (15) and ompP (25) on plasmid F (29). OmpP and OmpT have 87% sequence identity (25).…”

Section: Phop/phoq Regulates Ompsmentioning

confidence: 99%

A PhoP-Regulated Outer Membrane Protease of Salmonella enterica Serovar Typhimurium Promotes Resistance to Alpha-Helical Antimicrobial Peptides

Guina¹,

et al. 2000

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The outer membrane protein contents of Salmonella enterica serovar Typhimurium strains with PhoP/PhoQ regulon mutations were compared by two-dimensional gel electrophoresis. At least 26 species of outer membrane proteins (OMPs) were identified as being regulated by PhoP/PhoQ activation. One PhoP/PhoQ-activated OMP was identified by semiautomated tandem mass spectrometry coupled with electronic database searching as PgtE, a member of the Escherichia coli OmpT and Yersinia pestis Pla family of outer membrane proteases. Salmonella PgtE expression promoted resistance to alpha-helical cationic antimicrobial peptides (␣-CAMPs). Strains expressing PgtE cleaved C18G, an 18-residue ␣-CAMP present in culture medium, indicating that protease activity is likely to be the mechanism of OmpT-mediated resistance to ␣-CAMPs. PhoP/PhoQ did not regulate the transcription or export of PgtE, indicating that another PhoP/PhoQ-dependent mechanism is required for PgtE outer membrane localization. PgtE is a posttranscriptionally regulated component of the PhoP/PhoQ regulon that contributes to Salmonella resistance to innate immunity.

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“…The cleavage of sequences containing dibasic residues has been shown to be important for the inactivation of antibiotic peptides and colicins, the proteolysis of bacterial membrane proteins in trans, and the degradation of recombinant proteins expressed in E. coli (2,4,11,12,20,25,26,29,30). Since the enzyme is a membrane protein, it fractionates with the insoluble fraction in cell lysates and copurifies with protein inclusion bodies (33).…”

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Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpT

McCarter

Stephens

Shoemaker³

et al. 2004

J Bacteriol

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OmpT is a surface protease of gram-negative bacteria that has been shown to cleave antimicrobial peptides, activate human plasminogen, and degrade some recombinant heterologous proteins. We have analyzed the substrate specificity of OmpT by two complementary substrate filamentous phage display methods: (i) in situ cleavage of phage that display protease-susceptible peptides by Escherichia coli expressing OmpT and (ii) in vitro cleavage of phage-displayed peptides using purified enzyme. Consistent with previous reports, OmpT was found to exhibit a virtual requirement for Arg in the P1 position and a slightly less stringent preference for this residue in the P1 position (P1 and P1 are the residues immediately prior to and following the scissile bond). Lys, Gly, and Val were also found in the P1 position. The most common residues in the P2 position were Val or Ala, and the P3 and P4 positions exhibited a preference for Trp or Arg. Synthetic peptides based upon sequences selected by bacteriophage display were cleaved very efficiently, with k cat /K m values up to 7.3 ؋ 10 6 M ؊1 s ؊1 . In contrast, a peptide corresponding to the cleavage site of human plasminogen was hydrolyzed with a k cat /K m almost 10 6 -fold lower. Overall, the results presented in this work indicate that in addition to the P1 and P1 positions, additional amino acids within a six-residue window (between P4 and P2) contribute to the binding of substrate polypeptides to the OmpT binding site.

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The plasmid F OmpP protease, a homologue of OmpT, as a potential obstacle to E. coli‐based protein production

Cited by 20 publications

References 26 publications

Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpP

Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpP

A PhoP-Regulated Outer Membrane Protease of Salmonella enterica Serovar Typhimurium Promotes Resistance to Alpha-Helical Antimicrobial Peptides

Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpT

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