Cellfree Translation

E. coli lysates containing coupled transcription-translation reaction reagents with continuous supplies of nucleotides, amino acids, and other essential components have been developed for cell-free translation.8 The system applied for rapid trans lation of simple soluble proteins can generate yields of tens of milligrams.9 Technology development has allowed the production of isotopically labeled proteins for nuclear magnetic resonance (NMR) and x-ray crystallography in cell-free translation systems.10 It is possible to apply the technology directly on polymerase chain reaction (PCR) fragments, which enables the analysis of many constructs in parallel.11 However, the cell-free translation system still has certain severe limitations. Expression of membrane proteins, and particularly GPCRs, has not been very successful.12

Attempts to overexpress the human p2 adrenergic receptor (p2 AR) resulted in extremely low binding activity compared to other recombinant systems (unpublished results, Dr. Mikako Shirouzu, Genomics Sciences Center, RIKEN Institute, Yokohama, Japan). Continuous technology development may result in a breakthrough; in particular, the cell-free system based on a wheat germ lysate may have the potential to be applied successfully to membrane proteins.13 The addition or co-expression of chaperon proteins may enhance the production of membrane proteins.

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