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.

Figure 1. IRES-mediated translation in eukaryotic cell-free systems.

LUC encoding expression constructs harboring different IRESs in the EasyXpress pIX3.0 vector backbone were investigated. Cell-free protein synthesis was performed in linked and coupled transcription-translation systems using (A) Sf21, (B) CHO and (C) K562 cell extracts. Vectors containing a specific IRES are indicated by the name of the IRES insert. In the case of the CrPV IGR IRES, the influence of an AUG-to-GCU mutation of the initiation codon was investigated. Reactions were performed at standard conditions in the absence of a cap analogue. Relative light units were measured using a LUC reporter assay and the corresponding yields of active LUC in µg/mL were calculated based on a calibration curve. Yields of active LUC were determined from three independent experiments and the corresponding standard deviations were calculated.

Figure 2. Evaluation of optimal Mg(OAc)₂ (A) and KOAc (B) concentration in coupled eukaryotic cell-free expression systems.

De novo synthesized LUC was monitored after 3 h of incubation at 27°C (Sf21 cell extract) and 33°C (CHO and K562 cell extracts), respectively. Cell-free protein synthesis was performed using the optimized vector containing the CrPV IGR IRES harboring an AUG-to-GCU mutation of the initiation codon in the EasyXpress pIX3.0 vector backbone. Protein yields were normalized to the reaction with the highest yield of active LUC ( = 100%) for each cell-free system. Yields of active LUC were determined from three independent experiments using a LUC reporter assay and the corresponding standard deviations were calculated.

Figure 3. CrPV IGR IRES-mediated translation in coupled eukaryotic cell-free expression systems.

A) Time course analysis of cell-free synthesized LUC within 4 h of incubation using optimized reaction conditions. B) Impact of the optimization process on CrPV IGR IRES-mediated protein synthesis. Reaction temperature, incubation time, template concentration and ion concentrations (Mg(OAc)₂ and KOAc) were adapted to CrPV IGR IRES-directed translation. C) Comparison of the IRES-independent translation at standard conditions and CrPV IGR IRES-dependent translation at optimized conditions in coupled eukaryotic cell-free systems. Translations were carried out in the presence of 0.33 mM m⁷GpppG cap analogue. D) CrPV IGR IRES-mediated translation in a wheat germ CECF system within 24 h of incubation at 24°C. 100 mM KOAc was added to each dialysis reaction. Relative light units were measured using a LUC reporter assay and the corresponding yields of active LUC in µg/mL were calculated based on a calibration curve. Yields of active LUC were determined from three independent experiments and the corresponding standard deviations were calculated.

Figure 4. Autoradiographic analysis of CrPV IGR IRES-mediated translation in coupled eukaryotic cell-free systems.

The influence of the CrPV IGR IRES on protein expression of the secreted protein Mel-eYFP (29 kDa; A) as well as the membrane proteins Mel-Hb-EGF-eYFP (51 kDa; B) and Mel-EGFR-eYFP (162 kDa; C) was investigated in coupled systems based on lysates from Sf21, CHO and K562 cells. Plasmids encoding the target protein were equipped with (+) or without (−) the CrPV IGR IRES (GCU) in the EasyXpress pIX3.0 vector backbone. ¹⁴C-leucine-labeled, de novo synthesized proteins were visualized by autoradiography after gel electrophoresis.

Figure 5. CLSM analysis of eYFP-tagged proteins synthesized in coupled Sf21, CHO and K562 cell-free expression systems.

CLSM images depict the de novo synthesized secreted protein Mel-eYFP (A) as well as the membrane proteins Mel-Hb-EGF-eYFP (B) and Mel-EGFR-eYFP (C). Plasmids encoding the target proteins were equipped with (+) or without (−) the CrPV IGR IRES (GCU). In the case of Mel-eYFP, fluorescent vesicles indicate the translocation of the target protein into the lumen of the endogenous microsomes present in cell-free systems based on cultured Sf21, CHO and K562 cells. In the case of Mel-Hb-EGF-eYFP and Mel-EGFR-eYFP, microsomes show a fluorescent membrane due to the insertion of de novo synthesized membrane proteins. eYFP was excited at 488 nm and fluorescence emission was recorded with a long-pass filter in the wavelength range above 505 nm (LSM 510 Meta microscope, Zeiss).

Figure 6. Fluorescence analysis of eYFP-tagged proteins synthesized in coupled Sf21, CHO and K562 cell-free expression systems.

The image depicts the de novo synthesized secreted protein Mel-eYFP as well as the membrane proteins Mel-Hb-EGF-eYFP and Mel-EGFR-eYFP. Plasmids encoding the target proteins were equipped with (+) or without (−) the CrPV IGR IRES (GCU). Numbers indicate the increase of the expression levels using the CrPV IGR IRES-based construct compared to the no IRES control. No template controls were prepared in the same way as the samples, but instead of a DNA template, RNase-free water was added to the reaction. Samples were analyzed using the phosphorimager system (Typhoon TRIO+ Imager, GE Healthcare).

Figure 7. Expression of the glycoprotein EPO in prokaryotic and eukaryotic cell-free systems.

Eukaryotic expression was performed using the EasyXpress pIX3.0 vector equipped with the CrPV IGR IRES (GCU). In the case of the K562-based cell-free expression, EPO is presented before (−) and after (+) deglycosylation with PNGase F. For prokaryotic expression, the native signal peptide of EPO was replaced by the melittin signal sequence and cell-free expression of the target protein was operated using the EasyXpress pIX2.0 vector. ¹⁴C-leucine-labeled, de novo synthesized EPO (21 kDa, non-glycosylated) was visualized by autoradiography after gel electrophoresis.