Cell-free expression

Kahn TW, Beachy RN, Falk MM (1997) Cell-free expression of a GFP fusion protein allows quantitation in vitro and in vivo. Curr Biol 7 R207-R208... 

Klammt, C., Schwarz, D., Lohr, F. etal. (2006) Cell-free expression as an emerging technique for the large scale production of integral membrane protein. FEBS Journal, 273 (18), 4141—4153. 

Staunton D, Schlinkert R, Zanetti G, Colebrook SA, Campbell LD (2006) Cell-free expression and selective isotope labelling in protein NMR. Magn Reson Chem 44 S2-9... 

Klammt, C., Schwarz, D., Fendler, K., Haase, W., Dotsch, V. and Bernhard, F. Evaluation of detergents for the soluble expression of a-helical and ft-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system. FEBS J 272 6024-6038, 2005. 

Fig.1. The in vitro site-directed suppression mutagenesis system. Utilizing site-directed mutagenesis a specific codon within a gene under the control of an inducible promoter is converted to an amber termination codon. The gene is added to a cell-free expression system and transcription is induced in the presence of an aminoacyl suppressor tRNA, yielding protein containing the noncoded amino acid at the site corresponding to the termination codon...

The tag sequence is amplified from plasmid pTA-His that provides a flexible linker and a double (His)g tag. The two amplicons can then be assembled and amplified by using an upstream T7 primer containing the promoter plus Kozak sequence and start codon for translation. A T-term/ for primer is used downstream to amplify across the tag and linker. The resulting construct contains the gene, the promoter, and the tag (Figure 6.26). This is key to the technology for the construct is then used for cell-free expression of the protein. 

He, M. and Taussig, M.J., Single step generation of protein arrays from DNA by cell-free expression and in situ immobilization (PISA method). Nucleic Acid Res., 29(15), 1-6, 2001. 

E. coli cell-free expression system encapsulated in a phospholipid vesicle, which was transferred into a feeding solution containing ribonucleotides and amino acids. 

The Wheat Germ Cell-Free Expression System... 

Fig. 5. (Opposite page) Pictorial representation of GenDecoder specialized for functional analysis of genetic information. (A) Flow chart of cell-free expression process. (B) A photograph of GenDecoder, dimensions 1.5 m (W), 0.85 m (D), and 1.8 m (H). The robot is equipped with three robotic arms for pipetting and plate transfer, one incubator for transcription and translation, maximum capacity four plates, and centrifuge for mRNA recovery after ethanol precipitation. Incubator capacity can be increased by handling plates manually. (C) Proteins in 1 pL from each translated mixture were analyzed as in Fig. 4, thus 0.2 pL of original translational mix. (Reprinted from ref. 18 with kind permission of Springer Science and Business Media.)...

Endo, Y. and Sawasaki, T. (2004) High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system. J. Struct. Funct. Genomics 5(1-2), 45-57. 

Fig. 3. Schematic drawing of the pEU3b cell-free expression plasmid.

The third approach used mammalian sodium pumps expressing the a 3 and 3 1 subunits synthesized by in vitro translation in a cell-free expression system and then ineorporated into a planar phospholipid bilayer, where exposure to palytoxin elicited single-ehannel eurrents that had a conductance of about 10 pS (Hirsh and Wu 1997). 

In another study, proteins containing both mono- and diglycosylated amino acids, including glycosylated serine and tyrosine moieties, have been obtained by the suppression of nonsense codons in a cell-free expression system through the application of misacylated suppressor tRNAs activated with glycosylated serine and tyrosine derivatives (O Fig. 3) [32]. 

Cell-free expression of protein containing a glycosylated amino acid... 

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