Production of non‐glycosylated recombinant proteins in Nicotiana benthamiana plants by co‐expressing bacterial PNGase F

Abstract

SummaryApplication of tools of molecular biology and genomics is increasingly leading towards the development of recombinant protein-based biologics. As such, it is leading to an increased diversity of targets that have important health applications and require more flexible approaches for expression because of complex post-translational modifications. For example, Plasmodium parasites may have complex post-translationally modified proteins such as Pfs48 ⁄ 45 that do not carry N-linked glycans (Exp. Parasitol. 1998; 90, 165.) but contain potential N-linked glycosylation sites that can be aberrantly glycosylated during expression in mammalian and plant systems. Therefore, it is important to develop strategies for producing non-glycosylated forms of these targets to preserve biological activity and native conformation. In this study, we are describing in vivo deglycosylation of recombinant N-glycosylated proteins as a result of their transient co-expression with bacterial PNGase F (Peptide: N-glycosidase F). In addition, we show that the recognition of an in vivo deglycosylated plantproduced malaria vaccine candidate, Pfs48F1, by monoclonal antibodies I, III and V raised against various epitopes (I, III and V) of native Pfs48 ⁄ 45 of Plasmodium falciparum, was significantly stronger compared to that of the glycosylated form of plant-produced Pfs48F1. To our knowledge, neither in vivo enzymatic protein deglycosylation has been previously achieved in any eukaryotic system, including plants, nor has bacterial PNGase F been expressed in the plant system. Thus, here, we report for the first time the expression in plants of an active bacterial enzyme PNGase F and the production of recombinant proteins of interest in a non-glycosylated form.

Evaluation of the Impact of Codon Optimization and N-Linked Glycosylation on Functional Immunogenicity of Pfs25 DNA Vaccines Delivered by In Vivo Electroporation in Preclinical Studies in Mice

“…An unglycosylated merozoite surface protein 1 (MSP-1) proved more efficient in monkey experiments when challenged with a lethal dose of P. falciparum (30). Our findings with Pfs25 are in contrast to those of the Pfs48/45 studies in Nicotiana benthamiana that suggest that aberrant N-linked glycosylation of Plasmodium proteins by mammalian posttranslational modification machinery is likely to be detrimental to immunological outcomes of the antigen (31). On the other hand, our findings align well with posttranslational modification studies with Plasmodium falciparum apical membrane antigen 1 (PfAMA-1) produced in Pichia pastoris, which showed that immunogenicity and functional responses to PfAMA-1 were not altered by posttranslational modifications (32).…”

In vivo deglycosylation of recombinant proteins in plants by co-expression with bacterial PNGase F

“…The expression level of deglycosylated Pfs48F1 co-expressed with PNGase F was about 50 mg/kg of fresh leaf biomass and solubility was about 95%. Deglycosylated Pfs48F1 had a similar size as the in vitro deglycosylated Pfs48F1, 1 suggesting that Pfs48F1 was enzymatically deglycosylated by PNGase F in vivo. The efficiency of Pfs48F1 in vivo deglycosylation was confirmed by the glycan detection and mass spectrometry analyses.…”

“…The bacterial PNGase F sequence encompassing 314 amino acids (the full length of the catalytically active protein without a signal sequence) was optimized for the expression in Nicotiana benthamiana plants, cloned into the plant expression vector pGRD4 18 and expressed in N. benthamiana plants with a FLAG tag. 1 The expression of ~36 kDa PNGase F was confirmed by Western blot analysis using an anti-FLAG monoclonal antibody (mAb) (Fig. 1A).…”

“…Recently, we have developed a strategy of enzymatic deglycosylation of proteins in vivo by co-introducing bacterial PNGase F via agroinfiltration followed by transient expression in plants. 1 Here, we summarize our work on this topic and its potential implications.…”

“…The efficiency of Pfs48F1 in vivo deglycosylation was confirmed by the glycan detection and mass spectrometry analyses. 1 Malaria is a vector-borne infectious disease caused by protozoan parasites. It is widespread in tropical and subtropical regions, including parts of the Americas, Asia, and Africa with over 300-500 million cases and more 1 million deaths each year from around the world.…”