Author:
Langlais Claudia,Guilleaume Birgit,Wermke Nadja,Scheuermann Tina,Ebert Lars,LaBaer Joshua,Korn Bernhard
Abstract
Abstract
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.
Publisher
Springer Science and Business Media LLC
Reference22 articles.
1. Brenner SE: A tour of structural genomics. Nature Reviews Genetics. 2001, 2 (10): 801-809. 10.1038/35093574.
2. Wiemann S, Bechtel S, Bannasch D, Pepperkok R, Poustka A: The German cDNA network: cDNAs, functional genomics and proteomics. J Struct Funct Genomics. 2003, 4 (2–3): 87-96. 10.1023/A:1026148428520.
3. Imanishi T, Itoh T, Suzuki Y, O'Donovan C, Fukuchi S, Koyanagi KO, Barrero RA, Tamura T, Yamaguchi-Kabata Y, Tanino M, et al: Integrative annotation of 21,037 human genes validated by full-length cDNA clones. Plos Biology. 2004, 2 (6): 856-875. 10.1371/journal.pbio.0020162.
4. Bussow K, Scheich C, Sievert V, Harttig U, Schultz J, Simon B, Bork P, Lehrach H, Heinemann U: Structural genomics of human proteins – target selection and generation of a public catalogue of expression clones. Microbial Cell Factories. 2005, 4:
5. Hanning G, Makrides S: Strategies for optimising heterologous protein expression in Escherchia coli. TIBTECH. 1998, 16: 54-60.
Cited by
28 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献