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

Claudia Langlais,Nadja Wermke,Lars Ebert,Joshua Labaer,Birgit Guilleaume,Tina Scheuermann,Bernhard Korn

doi:10.1186/1472-6750-7-64

Abstract

BackgroundThe 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.ResultsIn 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%.ConclusionWe 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.

Highlights

The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies
We examined the effect of sequence optimisation on protein expression rate and protein yield by selecting randomly 87 out of the 960 open reading frames (ORFs) (Figure 1, Table 1) where protein expression had been unsuccessful in vivo or where transformation had failed in BL21(DE3)pLysS
We focused on a subset of 87 targets which had yielded no protein in E. coli in vivo

Summary

Introduction

The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. The use of E. coli has limitations, such as the aggregation of protein in insoluble inclusion bodies, problems with the expression of gene products toxic to the physiology of the host cell or proteolytic degradation of proteins in the cytoplasm [7] In light of these difficulties, cell-free expression systems are becoming increasingly popular [8,9,10,11,12,13,14]. The compatibility with PCR-generated templates as well as plasmids allows the in vitro expression reaction with E. coli extract to be optimised using silent mutations within PCR products [18] These sequence optimisations reduce unfavourable secondary structures in mRNA and improve the success rate of translation and protein expression. For cellfree protein expression with wheat germ lysate sequence optimisation is not necessary because of the eukaryotic nature of this source

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Conclusion