A NOVEL METHOD OF HIGH-YIELD CELL-FREE PROTEIN-SYNTHESIS

A novel method of cell-free protein synthesis named PER (preincubation followed by energy replenishment) mas developed. An incubation mixtur e containing wheat germ extract (WGE), ATP, GTP, acid amino acids was first preincubated at 30 degrees C for a certain time and then subject ed to centrifugal ultrafiltration to remove most of the low molecular weight components. After filtration, an energy replenishment buffer an d template mRNA were added to the filtrated incubation mixture and tra nslation was carried out at 30 degrees C for 1 h. Protein synthesis st arted earlier and progressed more rapidly using the PER method in comp arison with the conventional cell-free protein synthesis method: the r ate of protein production and the amount of protein produced increased 8-foId and 12-fold at maximum, respectively. For luciferase productio n using WGE, the optimal preincubation time at 30 degrees C was found to be 30 min. The presence of energy biochemicals in the incubation mi xture during preincubation significantly contributed to the increase i n protein productivity, presumably because (i) the formation of certai n complexes, such as the ternary complex (eIF-2.GTP.aminoacyl-tRNA) an d the 43S preinitiation complex (ternary complex.40S subunit), require s energy biochemicals; and (ii) the formation of the 43S preinitiation complex accelerates the formation of the 48S preinitiation complex (4 35 preinitiation complex. mRNA) which is a rate-limiting step in the i nitiation of protein synthesis. Filtration of the incubation mixture b y centrifugal ultrafiltration also played a major role in increasing t he protein productivity, presumably because formation of the complexes was accelerated by the temporary condensation that occurred during fi ltration. Energy replenishment (ER), supplied by a replenishment buffe r following ultrafiltration of the preincubated mixture, could restore the energy charge of the mixture to the level required for protein sy nthesis, The PER method can be used as a highly efficient cell-free pr otein 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.