High-throughput protein expression using cell-free system.
One of the main challenges in this post genomic era is the development and implementation of efficient methods of protein synthesis. A clear understanding of the role of genes in an organism is to comprehend the biological functions of all of its proteins. Acquiring this knowledge will depend in part on the success of rapid synthesis and purification of proteins. The future of structural genomics and functional proteomics depends on the availability of abundantly expressing, soluble proteins in a high-throughput manner. Conventional cell based methods of protein expression is rather laborious, time consuming and the ways to fail are numerous including solubility, toxicity to the host and instability (e.g. proteolysis). Cell-free or in vitro protein synthesis, on the other hand allows the expression and analysis of protein synthesis, may solve many of these problems. It is a simple open system which lends itself for manipulations and modifications to influence protein folding, disulfide bond formation, incorporation of unnatural amino acids, protein stability (by incorporating protease inhibitors in the system) and even the expression of toxic proteins. Cell-free synthesis can also be used as a reliable screening methodology for subsequent protein expression in vivo. Furthermore, this technology is readily amenable to automation. Here, we present a protocol for expressing recombinant proteins with high yield in a standard 96-well plate format using E. coli cell-free extract in a batch mode.
Sitaraman
Kalavathy
K
Protein Expression Laboratory, SAIC-Frederick, Inc., National Cancer Institute at Frederick, MD, USA.
Chatterjee
Deb K
DK
D000818:Animals; D002021:Buffers; D002458:Cell Fractionation / Q000379:methods*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D054680:Ribosome Subunits, Small, Bacterial / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic
D002021:Buffers; D011994:Recombinant Proteins; D004247:DNA