Roles of DegP in Prevention of Protein Misfolding in the Periplasm upon Overexpression of Penicillin Acylase in
            <i>Escherichia coli</i>

Pan, Kao-Lu; Hsiao, Hsu-Chou; Weng, Chiao-Ling; Wu, Mingqiu; Chou, C. Perry

doi:10.1128/jb.185.10.3020-3030.2003

Cited by 50 publications

(55 citation statements)

References 48 publications

(66 reference statements)

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“…In fact, the coexpression of DegP with overexpressed PhoA improved the secretion of this enzyme. Likewise, the enhancement of the production of soluble recombinant penicillin acylase in E. coli via coexpression of DegP has also been demonstrated (20,29).…”

Section: Discussionmentioning

confidence: 99%

Roles of Periplasmic Chaperone Proteins in the Biogenesis of Serine Protease Autotransporters of Enterobacteriaceae

et al. 2009

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The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent a large family of virulence factors. The prevailing model for autotransporter secretion comprises entry to the periplasm via the Sec apparatus, followed by an obscure series of steps in which the C terminus of the periplasmic species inserts into the outer membrane as a ␤-barrel protein, accompanied by translocation of the passenger domain to the bacterial cell surface. Little is known about the fate of the autotransporter proteins in the periplasm, including whether accessory periplasmic proteins are involved in translocation to the external milieu. Here we studied the role of the major periplasmic chaperones in the biogenesis of EspP, a prototype SPATE protein produced by Escherichia coli O157:H7. The yeast two-hybrid approach, secretion analysis of chaperone mutant strains, and surface plasmon resonance analysis (SPR) revealed direct protein-protein interactions between the periplasmic SurA and DegP chaperones and either the EspP-␤ or EspP passenger domains. The secretion of EspP was moderately reduced in the surA and skp mutant strains but severely impaired in the degP background. Site-directed mutagenesis of highly conserved aromatic amino acid residues in the SPATE family resulted in ϳ80% reduction of EspP secretion. Synthetic peptides containing aromatic residues derived from the EspP passenger domain blocked DegP and SurA binding to the passenger domain. SPR suggested direct proteinprotein interaction between periplasmic chaperones and the unfolded EspP passenger domain. Our data suggest that translocation of AT proteins may require accessory factors, calling into question the moniker "autotransporter."Secretion of proteins to the surface of gram-negative bacteria requires passage through the inner membrane (IM), the periplasm, and the outer membrane (OM). This formidable series of obstacles can be overcome only by complex biological processes. The autotransporter (AT) system, probably the most common gram-negative secretion mechanism (13), is characterized by formation of an OM ␤-barrel comprised of the C terminus of the periplasmic species. The precise events required for AT translocation across the OM, however, are controversial. The original model for OM translocation comprised targeting to the periplasm via the Sec apparatus, followed by formation of an OM ␤-barrel, which mediates passage of an unfolded or partially folded N-terminal passenger domain to the extracellular milieu (30). Three models of AT translocation have gained some acceptance (3, 16). According to the hairpin model, translocation of the passenger domain is initiated with the C-terminal end of the passenger forming a hairpin structure inside the AT ␤-barrel, followed by movement of the rest of the passenger through the barrel's pore in a C-to-N direction. Under the Omp85 model, the pore-forming Omp85 (YaeT in Escherichia coli) OM protein (OMP) facilitates insertion of the AT translocator domain into the OM, whereupon the AT passenger domain translocates th...

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Section: Discussionmentioning

confidence: 99%

Roles of Periplasmic Chaperone Proteins in the Biogenesis of Serine Protease Autotransporters of Enterobacteriaceae

et al. 2009

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“…Though the protease activity of DegP was shown to be primarily linked with the improvement (23), the possible contribution of the DegP chaperone activity could not be completely excluded. The results suggested that DegP could suppress the physiological toxicity in at least two ways (23). First, it degraded or refolded various abnormal periplasmic proteins generated upon pac overexpression.…”

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confidence: 93%

“…We previously demonstrated that the presence of exogenous DegP significantly reduced the amount of periplasmic proPAC inclusion bodies and enhanced the production of recombinant PAC in E. coli (19,23). Though the protease activity of DegP was shown to be primarily linked with the improvement (23), the possible contribution of the DegP chaperone activity could not be completely excluded.…”

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confidence: 99%

“…Using penicillin acylase (PAC), an important industrial enzyme for the production of many ␤-lactam antibiotics (12,27), as the target protein, strategies for enhancing recombinant protein production in E. coli have been developed in our lab (7,8,19,23). Formation of mature PAC in the periplasm of E. coli involves a series of posttranslational steps, including translocation and periplasmic processing/folding steps, which are unique for prokaryotic proteins (Fig.…”

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confidence: 99%

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Chaperone-Mediated Folding and Maturation of the Penicillin Acylase Precursor in the Cytoplasm of Escherichia coli

Weng

Narayanan

et al. 2005

Appl Environ Microbiol

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Expression of the leaderless pac gene (LL pac), which lacks the coding region for the signal peptide of penicillin acylase (PAC), in Escherichia coli was conducted. It was demonstrated that the PAC precursor, proPAC, can be produced and even processed to form mature PAC in the cytoplasm, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E. coli. The outcome of proPAC folding and PAC maturation could be affected by several factors, such as inducer type, proPAC formation rate, and chaperone availability. Misfolding of proPAC in the cytoplasm could be partially resolved through the coexpression of cytoplasmic chaperones, such as trigger factor, GroEL/ES, or DnaK/J-GrpE. The three chaperones tested showed different extents of the effect on proPAC solublization and PAC maturation, and trigger factor had the most prominent one. However, the chaperone-mediated solublization of proPAC did not guarantee its maturation, which is usually limited by the first autoproteolytic step. It was observed that arabinose could act as an effective inducer for the induction of LL pac expression regulated by the lac-derived promoter system of trc. In addition, PAC maturation could be highly facilitated by arabinose supplementation and coexpression of trigger factor, suggesting that the coordination of chaperone systems with proper culture conditions could dramatically impact recombinant protein production. This study suggests that folding/misfolding of proPAC could be a major step limiting the overproduction of PAC in E. coli and that the problem could be resolved through the search for appropriate chaperones for coexpression. It also demonstrates the analogy in the issues of proPAC misfolding as well as the expression bottleneck occurring in the cytoplasm (i.e., LL pac expression) and those occurring in the periplasm (i.e., wild-type pac expression).

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“…When unfolded proteins are accumulated in the periplasm, DegP is induced to clean the periplasm. DegP has also been reported to exhibit chaperone activity (17). However, it is not clear why only NlpE among the lipoproteins induces DegP.…”

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confidence: 98%

Effects of Lipoprotein Overproduction on the Induction of DegP (HtrA) Involved in Quality Control in the Escherichia coli Periplasm

Miyadai

Tanaka-Masuda

Matsuyama

et al. 2004

Journal of Biological Chemistry

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Roles of DegP in Prevention of Protein Misfolding in the Periplasm upon Overexpression of Penicillin Acylase in Escherichia coli

Cited by 50 publications

References 48 publications

Roles of Periplasmic Chaperone Proteins in the Biogenesis of Serine Protease Autotransporters of Enterobacteriaceae

Roles of Periplasmic Chaperone Proteins in the Biogenesis of Serine Protease Autotransporters of Enterobacteriaceae

Chaperone-Mediated Folding and Maturation of the Penicillin Acylase Precursor in the Cytoplasm of Escherichia coli

Effects of Lipoprotein Overproduction on the Induction of DegP (HtrA) Involved in Quality Control in the Escherichia coli Periplasm

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