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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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Wheat germ expression in vitro. Presented are western blots of 8 targets expressed with C-terminal (left) and N-terminal (right) 6xHis-tag. ORF Nr.: clone identifier. GUS: glucuronidase is the positive control. Successful protein expression was defined for values 2 – 4 and unsuccessful protein expression for values of 0 and 1. Bands of theexpected size are marked with a +.
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Figure 5: Wheat germ expression in vitro. Presented are western blots of 8 targets expressed with C-terminal (left) and N-terminal (right) 6xHis-tag. ORF Nr.: clone identifier. GUS: glucuronidase is the positive control. Successful protein expression was defined for values 2 – 4 and unsuccessful protein expression for values of 0 and 1. Bands of theexpected size are marked with a +.

Mentions: The aim of this experiment was to elucidate whether the wheat germ system would show an increase in the success rate and protein yield of the 87 selected open reading frames compared to the optimised in vitro expressions in E. coli. Two wild type PCR constructs were made for each open reading frame, one for production of a protein with a C-terminal 6xHis-tag and another for a protein with a N-terminal 6xHis-tag (Figure 5). A total of 75 proteins could be expressed in wheat germ lysate with either a C- or a N-terminal 6xHis-tag (86%, Figure 3). Out of the 16 open reading frames which were not expressed in the E. coli systems, 10 were now successfully expressed using wheat germ lysate (Table 2). However, 6 open reading frames did not express in the wheat germ system, but were previously successfully expressed in vitro in E. coli (Table 2). On average, based on western blotting analyses, the protein yield was higher in the wheat germ compared to expressions in the E. coli in vitro system, for identical human ORFs.


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)

Wheat germ expression in vitro. Presented are western blots of 8 targets expressed with C-terminal (left) and N-terminal (right) 6xHis-tag. ORF Nr.: clone identifier. GUS: glucuronidase is the positive control. Successful protein expression was defined for values 2 – 4 and unsuccessful protein expression for values of 0 and 1. Bands of theexpected size are marked with a +.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Wheat germ expression in vitro. Presented are western blots of 8 targets expressed with C-terminal (left) and N-terminal (right) 6xHis-tag. ORF Nr.: clone identifier. GUS: glucuronidase is the positive control. Successful protein expression was defined for values 2 – 4 and unsuccessful protein expression for values of 0 and 1. Bands of theexpected size are marked with a +.
Mentions: The aim of this experiment was to elucidate whether the wheat germ system would show an increase in the success rate and protein yield of the 87 selected open reading frames compared to the optimised in vitro expressions in E. coli. Two wild type PCR constructs were made for each open reading frame, one for production of a protein with a C-terminal 6xHis-tag and another for a protein with a N-terminal 6xHis-tag (Figure 5). A total of 75 proteins could be expressed in wheat germ lysate with either a C- or a N-terminal 6xHis-tag (86%, Figure 3). Out of the 16 open reading frames which were not expressed in the E. coli systems, 10 were now successfully expressed using wheat germ lysate (Table 2). However, 6 open reading frames did not express in the wheat germ system, but were previously successfully expressed in vitro in E. coli (Table 2). On average, based on western blotting analyses, the protein yield was higher in the wheat germ compared to expressions in the E. coli in vitro system, for identical human ORFs.

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