Dataset Information

Strategy for successful expression of the Pseudomonas putida nitrile hydratase activator P14K in Escherichia coli.

ABSTRACT: BACKGROUND: Activators of Nitrile hydratase (NHase) are essential for functional NHase biosynthesis. However, the activator P14K in P. putida is difficult to heterogeneously express, which retards the clarification of the mechanism of P14K involved in the maturation of NHase. Although a strep tag containing P14K (strep-P14K) was over-expressed, its low expression level and low stability affect the further analysis. RESULTS: We successfully expressed P14K through genetic modifications according to N-end rule and analyzed the mechanism for its difficult expression. We found that mutation of the second N-terminal amino-acid of the protein from lysine to alanine or truncating the N-terminal 16 amino-acid sequence resulted in successful expression of P14K. Moreover, fusion of a pelB leader and strep tag together (pelB-strep-P14K) at the N-terminus increased P14K expression. In addition, the pelB-strep-P14K was more stable than the strep-P14K. CONCLUSIONS: Our results are not only useful for clarification of the role of P14K involved in the NHase maturation, but also helpful for heterologous expression of other difficult expression proteins.

SUBMITTER: Liu Y

PROVIDER: S-EPMC3680314 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Strategy for successful expression of the Pseudomonas putida nitrile hydratase activator P14K in Escherichia coli.

Liu Yi Y Cui Wenjing W Fang Yueqin Y Yu Yuechun Y Cui Youtian Y Xia Yuanyuan Y Kobayashi Michihiko M Zhou Zhemin Z

BMC biotechnology 20130603

<h4>Background</h4>Activators of Nitrile hydratase (NHase) are essential for functional NHase biosynthesis. However, the activator P14K in P. putida is difficult to heterogeneously express, which retards the clarification of the mechanism of P14K involved in the maturation of NHase. Although a strep tag containing P14K (strep-P14K) was over-expressed, its low expression level and low stability affect the further analysis.<h4>Results</h4>We successfully expressed P14K through genetic modification ...[more]

PMID: 23731949

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Project description:Escherichia coli, commonly used in chemotaxis studies, is attracted mostly by amino acids, sugars, and peptides. We envisioned modifying the chemotaxis specificity of E. coli by expressing heterologous chemoreceptors from Pseudomonas putida enabling attraction either to toluene or benzoate. The mcpT gene encoding the type 40-helical bundle (40H) methyl-accepting chemoreceptor for toluene from Pseudomonas putida MT53 and the pcaY gene for the type 40H receptor for benzoate and related molecules from P. putida F1 were expressed from the trg promoter on a plasmid in motile wild-type E. coli MG1655. E. coli cells expressing McpT accumulated in chemoattraction assays to sources with 60 to 200 ?M toluene, although less strongly than the response to 100 ?M serine, but statistically significantly stronger than that to sources without any added attractant. An McpT-mCherry fusion protein was detectably expressed in E. coli and yielded weak but distinguishable membranes and polar foci in 1% of cells. E. coli cells expressing PcaY showed weak attraction to 0.1 to 1 mM benzoate, but 50 to 70% of cells localized the PcaY-mCherry fusion to their membrane. We conclude that implementing heterologous receptors in the E. coli chemotaxis network is possible and, upon improvement of the compatibility of the type 40H chemoreceptors, may bear interest for biosensing.IMPORTANCE Bacterial chemotaxis might be harnessed for the development of rapid biosensors, in which chemical availability is deduced from cell accumulation to chemoattractants over time. Chemotaxis of Escherichia coli has been well studied, but the bacterium is not attracted to chemicals of environmental concern, such as aromatic solvents. We show here that heterologous chemoreceptors for aromatic compounds from Pseudomonas putida at least partly functionally complement the E. coli chemotaxis network, yielding cells attracted to toluene or benzoate. Complementation was still inferior to native chemoattractants, like serine, but our study demonstrates the potential for obtaining selective sensing for aromatic compounds in E. coli.

Dataset Information

Strategy for successful expression of the Pseudomonas putida nitrile hydratase activator P14K in Escherichia coli.

Publications

Strategy for successful expression of the Pseudomonas putida nitrile hydratase activator P14K in Escherichia coli.

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