A novel method of high yield cell-free protein synthesis

Abstract

A novel method of cell-free protein synthesis named PER (preincubation followed by energy replenishment) was developed. An incubation mixture containing wheat germ extract (WGE), ATP, GTP, and amino acids was first preincubated at 30°C for a certain time and then subjected to centrifugal ultrafiltration to remove most of the low molecular weight components. After filtration, an energy replenishment buffer and template mRNA were added to the filtrated incubation mixture and translation was carried out at 30°C for 1 h. Protein synthesis started earlier and progressed more rapidly using the PER method in comparison with the conventional cell-free protein synthesis method: the rate of protein production and the amount of protein produced increased 8-fold and 12-fold at maximum, respectively. For luciferase production using WGE, the optimal preincubation time at 30°C was found to be 30 min. The presence of energy biochemicals in the incubation mixture during preincubation significantly contributed to the increase in protein productivity, presumably because (i) the formation of certain complexes, such as the ternary complex (eIF-2 · GTP · aminoacyl-tRNA) and the 43S preinitiation complex (ternary complex · 40S subunit), requires energy biochemicals; and (ii) the formation of the 43S preinitiation complex accelerates the formation of the 48S preinitiation complex (43S preinitiation complex · mRNA) which is a rate-limiting step in the initiation of protein synthesis. Filtration of the incubation mixture by centrifugal ultrafiltration also played a major role in increasing the protein productivity, presumably because formation of the complexes was accelerated by the temporary condensation that occurred during filtration. Energy replenishment (ER), supplied by a replenishment buffer following ultrafiltration of the preincubated mixture, could restore the energy charge of the mixture to the level required for protein synthesis. The PER method can be used as a highly efficient cell-free protein synthesis method for producing protein. ER is also a good method in itself for use in experimental studies on the mechanism of protein synthesis since it lessens the effects of energy charge decline.