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Open Access Methodology article

A set of ligation-independent in vitro translation vectors for eukaryotic protein production

Viola Bardóczy1, Viktória Géczi1, Tatsuya Sawasaki2, Yaeta Endo2 and Tamás Mészáros3*

Author Affiliations

1 Budapest University of Technology and Economics, Department of Applied Biotechnology and Food Science, 1111 Budapest, Mûegyetem rkp. 3., Hungary

2 Cell-Free Science and Technology Research Center, Ehime University, Bunkyo-cho 3-ban, Matsuyama 790-8577, Japan

3 Pathobiochemistry Research Group of Hungarian Academy of Sciences and Semmelweis University, 1088 Budapest, Puskin u. 9., Hungary

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BMC Biotechnology 2008, 8:32  doi:10.1186/1472-6750-8-32

Published: 27 March 2008

Abstract

Background

The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags.

Results

We designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein.

Conclusion

Four newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.