Genetic manipulation to identify limiting steps and develop strategies for high‐level expression of penicillin acylase in Escherichia coli

Chou, C. Perry; Yu, Chih‐Chang; Tseng, Jen-Hung; Lin, Ming-I; Lin, Hua‐Kuo

doi:10.1002/(sici)1097-0290(19990505)63:3<263::aid-bit2>3.3.co;2-k

Cited by 18 publications

(52 citation statements)

References 26 publications

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“…pARDegP was the mutagenesis template, and PSA/APSA were the mutagenesis primers; they contain a silent mutation with an extra NarI site for screening purposes. pTrcKnPAC2902 contains the pac operon, whose transcription is regulated by the trc promoter (8). It has the pBR322 replication origin and is therefore compatible with various pAR3-derived plasmids carrying the pACYC184 replication origin.…”

Section: Methodsmentioning

confidence: 99%

“…The protein content of the pellet was analyzed as the insoluble fraction. All the analytical methods in this study, including microbiological screening of PAC-producing strains, PAC assay, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and immunological analysis (Western blotting), were performed as described previously (8). ␤-Galactosidase was assayed as described previously (26).…”

Section: Methodsmentioning

confidence: 99%

“…The periplasmic processing mechanism is known to consist of various proteolytic steps via intramolecular autoproteolysis (15,38). The formation of inclusion bodies, which are composed primarily of PAC precursors in the periplasm, was recently identified as an important obstacle to the overproduction of PAC in E. coli (8,36,43). Hence, efforts have been directed to reducing the number of periplasmic PAC inclusion bodies.…”

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confidence: 99%

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

et al. 2003

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Enhancement of the production of soluble recombinant penicillin acylase in Escherichia coli via coexpression of a periplasmic protease/chaperone, DegP, was demonstrated. Coexpression of DegP resulted in a shift of in vivo penicillin acylase (PAC) synthesis flux from the nonproductive pathway to the productive one when pac was overexpressed. The number of inclusion bodies, which consist primarily of protein aggregates of PAC precursors in the periplasm, was highly reduced, and the specific PAC activity was highly increased. DegP was a heat shock protein induced in response to pac overexpression, suggesting that the protein could possibly suppress the physiological toxicity caused by pac overexpression. Coexpression of DegP S210A , a DegP mutant without protease activity but retaining chaperone activity, could not suppress the physiological toxicity, suggesting that DegP protease activity was primarily responsible for the suppression, possibly by degradation of abnormal proteins when pac was overexpressed. However, a shortage of periplasmic protease activity was not the only reason for the deterioration in culture performance upon pac overexpression because coexpression of a DegP-homologous periplasmic protease, DegQ or DegS, could not suppress the physiological toxicity. The chaperone activity of DegP is proposed to be another possible factor contributing to the suppression.The well-known genetic information of Escherichia coli and successful applications of recombinant DNA technology make it possible for a variety of attempts with genetic engineering techniques to overproduce recombinant proteins. Upon performing the cultivation for recombinant protein production, there are two primary goals: high-cell-density cultivation and high-level gene expression. Culture performance can be optimized when the two goals are achieved simultaneously. Highcell-density culture can be obtained by fed-batch cultivation (48), in which concentrated medium is fed gradually into the bioreactor. The primary concern of this operation is developing an optimum feeding strategy, based on which cells can be maintained in a high-energy state for enhancing gene expression while cells are growing.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

et al. 2003

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“…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%

“…The periplasmic processing mechanism consists of various proteolytic steps, including the intramolecular autoproteolysis for the first cleavage of the PAC precursor (proPAC; at the junction between the connecting peptide and ␤ subunit) and the subsequent proteolyses for chopping the connecting peptide (18,28). The formation of inclusion bodies, which are primarily composed of proPAC in the periplasm, was recently identified as an important obstacle for the overproduction of PAC in E. coli (8,26,29).…”

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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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Novel strategy for efficient screening and construction of host/vector systems to overproduce penicillin acylase inEscherichia coli

Chou¹,

Yu²,

Lin³

et al. 1999

Biotechnol. Bioeng.

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Genetic manipulation to identify limiting steps and develop strategies for high‐level expression of penicillin acylase in Escherichia coli

Cited by 18 publications

References 26 publications

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

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

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

Novel strategy for efficient screening and construction of host/vector systems to overproduce penicillin acylase inEscherichia coli

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