An elastin-like polypeptide (ELP) fusion protein was expressed in a cell-free protein synthesis system, and the expression profile was analyzed quantitatively. By selective addition of specific amino acids constituting ELP molecules, the expression level of the ELP fusion protein was improved by 1.3-1.8 times as high as positive control. This result implies that the expression amount of long repetitive polypeptides, which was dramatically decreased in vivo system, can be compensated to a degree by adding repeatedly consumed amino acids in vitro system. Presented results demonstrate the potential of cell-free protein synthesis for high-throughput study of various repetitive polypeptides. (C) 2009 Elsevier Inc. All rights reserved.