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.

Optimized ReplicatingRenillaLuciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function

“…In the design of LucR.CrPV proviral construct, we chose to utilize a Met codon to initiate translation of nef in order to preserve the native N-terminus of the Nef protein; however, translation from an Ala codon is more efficient. 82 Nevertheless, we are in the process of transferring the 6ATRi-nef approach, as well as select alternative IRES elements, into different strains of T/F HIV-1 to define their performance in the context of different proviral backbones.…”

Functional Analysis of Membrane Proteins Produced by Cell-Free Translation

“…In order to increase the protein yields, plasmids can be designed with additional regulatory elements such as CrPV IRES etc. [6].…”

“…Cell-free system offers all the conveniences required for proper synthesis of MPs. This method offers a high degree of controllability and provides a completely open system allowing direct manipulation of the reaction conditions to optimize protein folding, disulfide bond formation, incorporation of noncanonical amino acids and the synthesis of toxic proteins [4][5][6][7][8][9]. In comparison to conventional cell-based systems, cell-free systems offer rapid protein synthesis, purification and functional analysis.…”

Cell-Free Synthesis Meets Antibody Production: A Review

“…Recently, cell-free translation systems based on CHO and human cell extracts (K562 cells) were introduced [110,111]. Comparable to the insect cell-free translation system, these lysates contain endogenous microsomal vesicles, enabling the N-linked glycosylation of de novo synthesized target proteins and the embedding of membrane proteins into microsomal membranes.…”

“…Until now, eukaryotic cell-free translation systems based on insect cell extracts, human cell extracts and lysates from CHO cells could not reach such high production yields, although significant improvements have been made in recent years [68,110,111]. Nevertheless, for the future we anticipate an increase in productivity as more advanced reaction formats [68] can be combined with an improved template design [111].…”