Cell-free protein expression

Until recently, cell-free protein expression (also sometimes erroneously named in vitro protein expression) did not exhibit the productivity required for preparation of NMR samples, especially considering the high cost of using isotopically labeled starting material. Rather, it was exclusively used as an analytical tool that served to verify correct cloning or to study promotor sites. Because of the very low yields, detection of the expressed product usually required incorporation of a radioactive label (usually via 35S-methionine). 

The RTS system includes two different technology platforms for cell-free protein expression as well as a number of tools for finding optimal conditions (Scheme 1.1). All expression systems use the T7-polymerase for transcription and an E. coli lyzate with reduced nuclease and protease activity for translation. The conditions are optimized for a coupled transcription/translation reaction so that the DNA can be directly used as the template. 

Scheme 1.1 From gene to protein structure via cell-free protein expression.

This example demonstrates how cell-free protein expression can be used to rapidly optimize the reaction conditions as well as to shorten the time for spectral assignments by producing individually labeled proteins. 

Angenendt P, Nyarsik L, Szaflarski W, Glokler J, Nierhaus KH, Lehrach H, Cahill DJ, Lueking A. Cell-free protein expression and functional assay in nanowell chip format. Anal Chem 2004 76 1844—1849. 

The second example deals with our contribution to the development of semi-synthetic minimal cells, that in the recent years has been very technical, but with important conceptual and physical spin-off, as we will shortly illustrate here. Cell-free protein expression is a well established procedure, bom more than forty years ago ° and the first studies... 

Chizzolini, F., Forlin, M., Cecchi, D., Mansy, S.S. Gene position more strongly influences cell-free protein expression from operons than T7 transcriptional promoter strength. ACS Synth. Biol. 3, 363-371 (2014)... 

Nucleic acid Ligation of protein gene with X-DNA Cell-free protein expression with high... 

Dittrich, P. S., M. Jahnz, and P. Schwille. 2005. A new embedded process for compartmentalized cell-free protein expression and on-line detection in microfluidic devices. ChemBioChem. 6 811-814. 

Beernink, P.T., Segelke, B.W., and Coleman, M.A., High-throughput, cell-free protein expression screening using the RTS 100 E. coli HY kit, Biochemica (Roche), 1,4, 2003. 

---