title: IRES-mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems.
abstract: Internal ribosome entry site (IRES) elements found in the 5' untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR) of the Cricket paralysis virus (CrPV) genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established systems.
authors.affiliation: Fraunhofer Institute for Biomedical Engineering (IBMT) Branch Potsdam-Golm, Potsdam, Germany.
authors.forename: Andreas K
authors.initials: AK
authors.lastname: Brödel
authors.affiliation: Fraunhofer Institute for Biomedical Engineering (IBMT) Branch Potsdam-Golm, Potsdam, Germany.
authors.forename: Andrei
authors.initials: A
authors.lastname: Sonnabend
authors.affiliation: Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom.
authors.forename: Lisa O
authors.initials: LO
authors.lastname: Roberts
authors.affiliation: Fraunhofer Institute for Biomedical Engineering (IBMT) Branch Potsdam-Golm, Potsdam, Germany.
authors.forename: Marlitt
authors.initials: M
authors.lastname: Stech
authors.affiliation: Fraunhofer Institute for Biomedical Engineering (IBMT) Branch Potsdam-Golm, Potsdam, Germany.
authors.forename: Doreen A
authors.initials: DA
authors.lastname: Wüstenhagen
authors.affiliation: Fraunhofer Institute for Biomedical Engineering (IBMT) Branch Potsdam-Golm, Potsdam, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kubick
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D002474:Cell-Free System; D005057:Eukaryotic Cells / Q000378:metabolism*; D005453:Fluorescence; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis*; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis*; D018613:Microscopy, Confocal; D014176:Protein Biosynthesis / Q000235:genetics*; D014780:Viruses / Q000235:genetics
chemical_list: D020121:5' Untranslated Regions; D006023:Glycoproteins; D008565:Membrane Proteins