IRES-mediated Pichia pastoris cell-free protein synthesis

Abstract

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. Graphical Abstract

Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α

“…By varying the 5′ cap structure or the initiation factors, the expression level can be optimally controlled [ 41 , 42 ]. In contrast, CrPV IRES-dependent initiation, which is often used in cell-free reactions, is more difficult to control or modify because it is independent of the cap structure and initiation factors [ 14 , 43 , 44 ]. A significant regulator of translational initiation is the initiation factor eIF2α.…”

“…Frequently, the CrPV IRES is utilized for translation initiation in eukaryotic cell-free systems because it is independent of the cap structure and translation initiation factors [ 49 , 50 ]. Although the Encephalomyocarditis virus (EMCV) IRES is commonly used in mammalian cell lines, its effectiveness in cell-free systems was poor [ 14 , 51 , 52 ]. The EMCV IRES operates independently of the cap structure, but unlike CrPV IRES, initiation factors such as eIF2 are required [ 53 ].…”

Engineering cell-free systems by chemoproteomic-assisted phenotypic screening

“…P. pastoris CFPS can yield 50-300 mg mL À1 of protein, with maximal yields in S. cerevisiae trailing at 20 mg mL À1 . [84][85][86] The latter has been obtained after significant efforts to boost yields and overall cost efficiency using numerous approaches. 30,50,60,87 Key strategies involved fine-tuning of extract metabolic processes through optimization of growth and harvesting conditions and elimination of proteins with inhibitory effects on CFPS such as PEP4 protease, XRN1 nuclease and STM1 ribosome inactivation factor.…”

A Cost-Effective Pichia pastoris Cell-Free System Driven by Glycolytic Intermediates Enables the Production of Complex Eukaryotic Proteins

“…In recent years, various protocols have been developed to establish cost-effective S. cerevisiae-based cell-free protein synthesis based on different cultivation, disruption, and lysate processing methods, as well as additives to the cell-free reaction [24][25][26]. Various protocols could be transferred to P. pastoris, and new methods could be established to use P. pastoris lysates in a cell-free format to synthesize, for instance, virus-like particles that are difficult to produce [27][28][29].…”