An increased rate of cell-free protein synthesis by condensing wheat-germ extract with ultrafiltration membranes

Abstract

Wheat-germ extract for cell-free protein synthesis was condensed with ultrafiltration membranes of which the molecular cut-off values were 10 kDa, 100 kDa, and 300 kDa. Reaction conditions of the cell-free system were optimized for the condensed extracts, which needed a higher concentration of creatine phosphate than the uncondensed one, probably due to the increased activity of degradation of ATP and GTP. By using the condensed extract and optimized reaction conditions, the rate of protein synthesis was increased 2- to 3-fold compared with using an uncondensed extract, and about 10-fold compared with conventional conditions. Condensation of the extract with the 300-kDa membrane showed the highest productivity, which was about 30 micrograms dihydrofolate reductase protein ml-1 h-1. The final amount of synthesized protein was one third of that of a continuous-flow cell-free (CFCF) system reported by Endo et al. [J. Biotechnol., 25, 221-230 (1992)] but the productivity was 5-fold higher than that obtained by the CFCF system.