IRES-mediated Pichia pastoris cell-free protein synthesis

Abstract Cell-free protein synthesis (CFPS) system is an ideal platform for fast and convenient research has been used macromolecular assembly, unnatural amino acid embedding, glycoprotein production, more. To realize the construction of efficient eukaryotic CFPS with advantages low cost short time, a based on yeast Pichia pastoris was built in this study. The internal ribosomal entry site (IRES) can independently initiate translation thus promote synthesis. Kozak sequences facilitate initiation. Therefore, screening IRES its combination performed, which cricket paralysis virus (CRPV) exhibited as best initiation element from 14 different IRESs. Furthermore, components reaction environment were explored. yield nearly doubled by addition RNase inhibitor. cell extract amount, energy regeneration (phosphocreatine phosphocreatine kinase), metal ions (K + Mg 2+ ) optimized to achieve yield. This P. extend platform, providing enabling technology prototyping design functional Graphical