Fusion of a highly N-glycosylated polypeptide increases the expression of ER-localized proteins in plants

Abstract

Plants represent promising systems for producing various recombinant proteins. One key area of focus for improving this technology is developing methods for producing recombinant proteins at high levels. Many methods have been developed to increase the transcript levels of recombinant genes. However, methods for increasing protein production involving steps downstream of transcription, including translation, have not been fully explored. Here, we investigated the effects of N-glycosylation on protein production and provide evidence that N-glycosylation greatly increases the expression levels of ER-targeted recombinant proteins. Fusion of the extracellular domain (M domain) of protein tyrosine phosphatase receptor type C (CD45), which contains four putative N-glycosylation sites to a model protein, leptin at the C-terminus, increased recombinant protein levels by 6.1 fold. This increase was specific to ER-targeted proteins and was dependent on N-glycosylation. Moreover, expression levels of leptin, leukemia inhibitory factor and GFP were also greatly increased by fusion of M domain at either the N or C-terminus. Furthermore, the increase in protein levels resulted from enhanced translation, but not transcription. Based on these results, we propose that fusing a small domain containing N-glycosylation sites to target proteins is a powerful technique for increasing the expression levels of recombinant proteins in plants.

Cost‐effective production of tag‐less recombinant protein in Nicotiana benthamiana

“…The final PCR products were digested with BamHI and XhoI restriction endonucleases and inserted into the plant expression vector 326‐GFP, digested with BamHI and XhoI (Kim et al ., ). The M domain, the N‐terminal fragment from amino acid positions 231 to 290 of human protein tyrosine phosphatase receptor type C (CD45), was fused to the N‐terminus of CBM3‐bdSUMO‐hIL6 by overlapping PCR using plasmid EelepfM (Kang et al ., ) as a template and the primer sets PF‐5, PR‐6, PF‐7 and PR‐8 (Table S1). The plasmid obtained by PCR amplification, named 326 ‐ M‐CBM3‐bdSUMO‐hIL6 , was digested with PstI and EcoRI and inserted into the plant expression binary vector pCAMBIA1300, digested with PstI and EcoRI , to give p1300‐C3bdSU‐hIL6 (Figure a).…”

“…A previous study found that the endoplasmic reticulum (ER) accumulates high levels of recombinant proteins (Gomord et al, 1997;Kang et al, 2018;Sohn et al, 2018;Wandelt et al, 1992). Thus, to achieve a high level of expression, we used the BiP leader sequence to target the recombinant protein to the ER.…”

“…We also included another important component, the highly mannosylated N-glycan-containing polypeptide (M) domain. The M domain, derived from human protein tyrosine phosphatase, receptor type C (CD45), has multiple N-glycosylation sites and, when fused to a target protein, increases translation levels up to sevenfold (Kang et al, 2018). The M domain was inserted next to the BiP leader sequence, so it could be removed from the target protein, together with CBM3 and bdSUMO, by cleavage with bdSENP1 protease (Figure 1a).…”

In Vivo Removal of N-Terminal Fusion Domains From Recombinant Target Proteins Produced in Nicotiana benthamiana

“…The ER localization renders recombinant proteins to be subject to posttranslational modification such as N-glycosylation and disulfide bond formation, which are critical for protein folding, stability, and functionality (Gomord and Faye, 2004). In addition, targeting of recombinant proteins to the ER significantly improved production yields in plants (Schouten et al, 1996;Nausch et al, 2012;Kang et al, 2018). Therefore, we first determined whether bdSENP1 was capable of cleaving its substrate in the ER in vivo.…”

“…Moreover, the fusion of fungal hydrophobins (HFBI) (Gutiérrez et al, 2013) and the prolinerich domain of g-zein (Conley et al, 2011), a maize seed storage protein, increase the accumulation of recombinant proteins in plants. Fusions of an N-glycosylation domain (M domain) derived from human CD45 to either the C-or N-terminus (Kang et al, 2018), and of SBA, a sugar-binding lectin from soybean (Alqazlan et al, 2019), have also recently been shown to greatly enhance expression of fusion proteins in plants.…”