Protein synthesis in cell-free reticulocyte lysates on multi-hour incubation

Abstract

Cell-free protein synthesis in rabbit reticulocyte lysate translation mixtures has been studied during multi-hour incubations. In an impaired lysate obtained from cells stored at 0'C before lysis, and showing a low initial rate of synthesis, translation could be stimulated by a factor of 4by including RNase inhibitor and additional ATP and GTP. In translation mixtures prepared from normal lysates, protein svnthesis could be improved by -50oh by the addition of excess GTP. The observed increases in protein synthesis were obtained by improved maintenance of the initial rate of synthesis.Index Entries: Cell-free protein synthesis; cell-free translation; reticulocyte lysate, and behavior on multi-hour incubation. INTRODqCTIONThe in vitro, cell-free synthesis of proteins is a much more difficult enterprise than in vivo synthesis using recombinant DNA technology, but it is a technique of real potential utility for the synthesis of proteins containing labeled or unnatural aminoacids, and for synthesis of proteins containing certain types of unnatural amino acid sequences. Chemical methods work well for the synthesis of relatively small peptides ( < 40 amino acid residues in length) but lose efficiency as synthesis of larger products is pursued (1,2). For larger proteins (MW>20,000), chemical synthesis may never be able to rival the combination of rDNA methods coupled with fermentation.

Transforming biochemical engineering with cell‐free biology

“…The initial and more developed cell-free technology is CFPS. E.coli bacterial cells (Swartz, 2006), red blood cells (Findeis and Whitesides, 1987), and wheat kernels (Sawasaki et al, 2002)) have all been used as the source of the protein synthesis catalysts. In all cases, the cells are lysed and the contents diluted.…”