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


CONCLUSIONS
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.
Langlais
Claudia
C
MRC Toxicology Unit, Protein Profiling Group, Hodgkin Building, Lancaster Road, Leicester, LE1 9HN, UK. cl40@leicester.ac.uk
Guilleaume
Birgit
B
Wermke
Nadja
N
Scheuermann
Tina
T
Ebert
Lars
L
LaBaer
Joshua
J
Korn
Bernhard
B
D002474:Cell-Free System / Q000378:metabolism*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D011994:Recombinant Proteins / Q000378:metabolism*
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins