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

Langlais C, Guilleaume B, Wermke N, Scheuermann T, Ebert L, LaBaer J, Korn B - BMC Biotechnol. (2007)

Bottom Line: Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Toxicology Unit, Protein Profiling Group, Hodgkin Building, Lancaster Road, Leicester, LE1 9HN, UK. cl40@leicester.ac.uk

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.

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E.coli expression in vitro. Presented are western blots of 8 targets expressed with C-terminal wild type, C-terminal mutant, N-terminal wild type and original plasmid DNA templates (from left to right). Successful protein expression was defined for values 2 – 4 and unsuccessful protein expression for values of 0 and 1. Bands of the expected size are marked with a +.
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Figure 4: E.coli expression in vitro. Presented are western blots of 8 targets expressed with C-terminal wild type, C-terminal mutant, N-terminal wild type and original plasmid DNA templates (from left to right). Successful protein expression was defined for values 2 – 4 and unsuccessful protein expression for values of 0 and 1. Bands of the expected size are marked with a +.

Mentions: Of these 87 samples, 37 samples (43%) were successfully expressed in vitro using the original plasmid DNA. After sequence optimisation, we increased the success rate of protein expression up to 74 samples (85%) in the cell-free E. coli system (Figure 3, 4).


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

Langlais C, Guilleaume B, Wermke N, Scheuermann T, Ebert L, LaBaer J, Korn B - BMC Biotechnol. (2007)

E.coli expression in vitro. Presented are western blots of 8 targets expressed with C-terminal wild type, C-terminal mutant, N-terminal wild type and original plasmid DNA templates (from left to right). Successful protein expression was defined for values 2 – 4 and unsuccessful protein expression for values of 0 and 1. Bands of the expected size are marked with a +.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2131746&req=5

Figure 4: E.coli expression in vitro. Presented are western blots of 8 targets expressed with C-terminal wild type, C-terminal mutant, N-terminal wild type and original plasmid DNA templates (from left to right). Successful protein expression was defined for values 2 – 4 and unsuccessful protein expression for values of 0 and 1. Bands of the expected size are marked with a +.
Mentions: Of these 87 samples, 37 samples (43%) were successfully expressed in vitro using the original plasmid DNA. After sequence optimisation, we increased the success rate of protein expression up to 74 samples (85%) in the cell-free E. coli system (Figure 3, 4).

Bottom Line: Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Toxicology Unit, Protein Profiling Group, Hodgkin Building, Lancaster Road, Leicester, LE1 9HN, UK. cl40@leicester.ac.uk

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.

Show MeSH
Related in: MedlinePlus