title: Characterizing IGR IRES-mediated translation initiation for use in yeast cell-free protein synthesis.
abstract: Eukaryotic cell-free protein synthesis (CFPS) systems are limited, in part, by inefficient translation initiation. Here, we report three internal ribosome entry site (IRES) sequences from the Dicistroviridae family that are highly active in yeast CFPS. These include the intergenic region (IGR) IRES from cricket paralysis virus (CrPV), plautia stali intestine virus (PSIV) and Solenopsis invicta virus 1 (SINV1). Optimization of combined transcription and translation (Tx/Tl) CFPS reactions primed with linear DNA containing the CrPV IGR IRES resulted in batch synthesis yields of 0.92 ± 0.17 μg/mL luciferase. Further template engineering, such as including the first 12 nt of native CrPV gene, increased yields to 2.33 ± 0.11 μg/mL. We next observed that the inclusion of a 50 nt poly(A) to the 3' end of the IGR IRES-mediated message increased yields an additional 81% to 4.33 ± 0.37 μg/mL, without any effect on mRNA stability or copy number. This was surprising because the CrPV IGR IRES requires no known translation initiation factors. Lastly, we investigated a method to inhibit background expression through competitive inhibition by supplying the reaction with 5' cap structure analog. This study highlights the crucial role translation initiation plays in yeast CFPS and offers a simple platform to study IRES sequences.
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Technological Institute, E136, Evanston, IL 60208-3120, USA; Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, IL 60208-3120, USA.
authors.forename: C Eric
authors.initials: CE
authors.lastname: Hodgman
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Technological Institute, E136, Evanston, IL 60208-3120, USA; Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, IL 60208-3120, USA; Member, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 676 North St Clair Street, Suite 1200, Chicago, IL 60611-3068, USA; Institute for BioNanotechnology in Medicine, Northwestern University, 303 East Superior Street, Suite 11-131, Chicago, IL 60611-2875, USA. Electronic address: m-jewett@northwestern.edu.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
mesh_terms: D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D005818:Genetic Engineering; D010442:Peptide Chain Initiation, Translational; D011061:Poly A / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D038621:Regulatory Sequences, Ribonucleic Acid; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014780:Viruses / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D014764:Viral Proteins; D011061:Poly A