IRES-mediated Pichia pastoris cell-free protein synthesis

Cell-free protein synthesis (CFPS) system is an ideal platform for fast and convenient protein research and has been used for macromolecular assembly, unnatural amino acid embedding, glycoprotein production, and more. To realize the construction of an efficient eukaryotic CFPS platform with the advantages of low cost and short time, a CFPS system based on the yeast Pichia pastoris was built in this study. The internal ribosomal entry site (IRES) can independently initiate translation and thus promote protein synthesis. The Kozak sequences can facilitate translation initiation. Therefore, the screening of IRES and its combination with Kozak was performed, in which cricket paralysis virus (CRPV) exhibited as the best translation initiation element from 14 different IRESs. Furthermore, the system components and reaction environment were explored. The protein yield was nearly doubled by the addition of RNase inhibitor. The cell extract amount, energy regeneration system (phosphocreatine and phosphocreatine kinase), and metal ions (K+ and Mg2+) were optimized to achieve the best protein synthesis yield. This P. pastoris CFPS system can extend the eukaryotic CFPS platform, providing an enabling technology for fast prototyping design and functional protein synthesis.

Automated summary

A cell-free protein synthesis (CFPS) system based on Pichia pastoris yeast was constructed, providing a low-cost and time-efficient platform for protein research and synthesis. The combination of IRES and Kozak sequences revealed that cricket paralysis virus (CRPV) was the best translation initiation element among 14 different IRESs. Optimization of system components and reaction environment led to increased protein yield. This P. pastoris CFPS system extends the eukaryotic CFPS platform and enables fast prototyping design and functional protein synthesis.