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High-level expression of soluble subunit b of F1F0 ATP synthase in Escherichia coli cell-free system

  • Biotechnologically Relevant Enzymes and Proteins
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Abstract

The overexpression of subunit b of F1F0 adenosine triphosphate (ATP) synthase from Escherichia coli is so toxic that it even prevents the transformation of plasmids encoding this protein into E. coli BL21 (DE3). In the present work, E. coli cell-free system was chosen as an alternative to express this highly toxic membrane protein. This protein was either produced as precipitates followed by detergent resolubilization or expressed as a soluble form with detergent addition. Among several types of tested detergents, Brij 58 could effectively solubilize approximately 85% of the target membrane protein within a wide range of concentration (48 to 178 times critical micelle concentration [CMC]) with little effect on the expression level. With the presence of Brij 58 at the final concentration of 96 times CMC in the E. coli cell-free system, 789 μg/mL of soluble subunit b was achieved after 4 h biosynthesis, which is the highest level for the expression of membrane proteins in a batch-mode cell-free expression system. The present work provides a rapid and efficient procedure of expressing one membrane protein with high cytotoxicity in the cell-free system and will be helpful to further exploration of reconstituting F1F0 ATP synthase into liposome or polymer vesicle to design a nanoelectromechanical system device.

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Acknowledgements

This work was financially supported by The National Natural Science Foundation of China (grant nos. 20736008 and 20676115) and The Ministry of Science and Technology (grant nos. 2007AA021702 and 2009CB918600), The People's Republic of China.

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Authors and Affiliations

  1. Institute of Bioengineering, Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou, 310027, China

    Jiazhang Lian, Yi Ma, Jin Cai, Ming Wu & Zhinan Xu

  2. School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, China

    Yi Ma, Jufang Wang & Xiaoning Wang

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  1. Jiazhang Lian
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  2. Yi Ma
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  3. Jin Cai
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  4. Ming Wu
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  5. Jufang Wang
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  6. Xiaoning Wang
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  7. Zhinan Xu
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Correspondence to Zhinan Xu.

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Lian, J., Ma, Y., Cai, J. et al. High-level expression of soluble subunit b of F1F0 ATP synthase in Escherichia coli cell-free system. Appl Microbiol Biotechnol 85, 303–311 (2009). https://doi.org/10.1007/s00253-009-2055-z

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  • DOI: https://doi.org/10.1007/s00253-009-2055-z

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