Cell-free synthesis of tachyplesin, an antimicrobial peptide from tachypleus tridentatus

Abstract

Tachyplesin is an antimicrobial peptide isolated from the he es of horseshoe crab (Tachypleus tridentatus). Due to the importance of producing highly active peptides that have great commercial application, we attempted to produce this protein using a continuously coupled transcription-translation in a wheat germ extract system. The system works by using a continuous flow of feeding solution containing nucleoside triphosphates and amino acids into a 1-ml reactor containing wheat germ extract, plasmid DNA, and transcription enzyme, and continuous removal of translation product through an ultrafiltration membrane fitted in the reactor. The amount of protein synthesized reached to about 80 mg after 27 h of continuous reaction. Autoradiogram of the translated product showed a distinct band at about 8.44 kDa corresponding to the calculated molecular weight of tachyplesin. Continuously coupled transcription-translation in a cell-free system offers many possible applications to create new products in biotechnology