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A systematic approach for testing expression of human full-length proteins in cell-free expression systems

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posted on 2016-04-13, 12:10 authored by Claudia Langlais, B. Guilleaume, N. Wermke, T. Scheuermann, L. Ebert, J. LaBaer, B. Korn
Background The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems. Results In a pre-screen, we evaluated the expression of 960 human full-length open reading frames in Escherichia coli (in vivo and in vitro). After analysing the protein expression rate and solubility, we chose a subset of 87 plasmids yielding no protein product in E. coli in vivo. These targets were subjected to a more detailed analysis comparing a prokaryotic cell-free E. coli system with an eukaryotic wheat germ system. In addition, we determined the expression rate, yield and solubility of those proteins. After sequence optimisation for the E. coli in vitro system and generating linear templates for wheat germ expression, the success rate of cell-free protein expression reached 93%. Conclusion We have demonstrated that protein expression in cell-free systems is an appropriate technology for the successful expression of soluble full-length proteins. In our study, wheat germ expression using a two compartment system is the method of choice as it shows high solubility and high protein yield.

Funding

This work was supported by Roche Diagnostics GmbH.

History

Citation

BMC Biotechnology20077:64

Version

  • VoR (Version of Record)

Published in

BMC Biotechnology20077:64

Publisher

BioMed Central

issn

1472-6750

eissn

1472-6750

Acceptance date

2007-10-03

Copyright date

2007

Available date

2016-04-13

Publisher version

http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-7-64

Language

en

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