IRES-Mediated Translation of Membrane Proteins and Glycoproteins in Eukaryotic Cell-Free Systems

Andreas K Brödel,Andrei Sonnabend,Marlitt Stech,Doreen A Wüstenhagen,Stefan Kubick,Lisa O Roberts,Eric Jan

doi:10.1371/journal.pone.0082234

Andreas K Brödel, Andrei Sonnabend + Show 5 more

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https://doi.org/10.1371/journal.pone.0082234

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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.

Highlights

Translation, the process of mRNA-encoded protein synthesis, can be subdivided into the phases of initiation, elongation, termination and ribosome recycling [1]
Highest yields of de novo synthesized LUC were obtained from expression constructs containing the Cricket paralysis virus (CrPV) intergenic region (IGR) internal ribosome entry site (IRES) in coupled cell-free platforms
As it is known that the majority of Dicistrovirus IGR IRESs display maximum efficiency when GCU is the initiation codon [26], we assessed the efficiency of this IRES in this context compared to an AUG codoncontaining IRES

Summary

Introduction

Translation, the process of mRNA-encoded protein synthesis, can be subdivided into the phases of initiation, elongation, termination and ribosome recycling [1]. Of these phases, translation initiation is the most complex and rate-limiting step [2], representing a major barrier in the activation of a cell-free protein synthesis reaction [3]. Recently developed eukaryotic cell-free expression systems have removed the requirement for this process by employing internal ribosome entry site (IRES) elements [7,8,9,10]. IRES elements are highly structured RNA sequences found within the 59 untranslated region (UTR) of viral genomes or cellular mRNAs that function to recruit ribosomes for the initiation of translation. Many IRES elements do not initiate protein synthesis in cell extracts from various organisms

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