METHOD OF PREPARING POLYPEPTIDES IN A CELL-FREE TRANSLATION SYSTEM

Polypeptides are synthesized over a several hour period in a cell-free system containing an exogenous prokaryotic RNA polymerase, a DNA molecule which encodes a polypeptide of interest, and additional substrates. In the cell-free polypeptide synthesis method, a mixture which includes a eukaryotic cell extract capable of supporting in vitro translation, ATP, GTP, CTP, UTP, and amino acids is added to a vessel containing an ultrafiltration membrane barrier. Reaction substrates including ATP, GTP, CTP, UTP and amino acids are continuously delivered to the vessel at a rate that maintains their initial concentration in the vessel while the products of the process, including AMP, GDP, CDP, UDP, pyrophosphate, inorganic phosphate, and the polypeptide product are continuously removed from the vessel through the ultrafiltration barrier. Included in the reaction mixture is an exogenous prokaryotic RNA polymerase and a DNA molecule which encodes the polypeptide of interest and contains a promoter site specific to the exogenous RNA polymerase.