In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus.

Tarlan Mamedov,Rifat Ungor,Gulnara Hasanova,Kader Cicek,Burcu Gulec,Renwick Dobson

doi:10.1371/journal.pone.0183589

Tarlan Mamedov, Rifat Ungor + Show 4 more

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https://doi.org/10.1371/journal.pone.0183589

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Journal: PloS one	Publication Date: Aug 21, 2017
Citations: 20	License type: CC BY 4.0

Affiliation: Akdeniz University

Abstract

A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F). Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-β-N-acetylglucosaminidase (EC3.2.1.96, Endo H), another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes.

Highlights

Plant based transient expression system is a promising technology for the production of various recombinant proteins including vaccine antigens, therapeutic proteins, antibodies and industrial enzymes
The ability of plants to glycosylate proteins can be a significant limitation for the production of proteins not carrying N-linked glycans in the native hosts such as Pfs48/45 protein of Plasmodium falciparum, the A chain of human factor XIII, or the protective antigen (PA) of Bacillus anthracis.These proteins are not glycoproteins in the native hosts, they contain potential N-linked glycosylation sites that can be aberrantly glycosylated during production in any eukaryotic expression system
Numerous studies in recent years demonstrated that plant based transient expression system has high expression capacity and provides safe, fast, inexpensive production of valuable recombinant proteins of interest within a short time frame [21,22,23,24,25]

Summary

Introduction

Plant based transient expression system is a promising technology for the production of various recombinant proteins including vaccine antigens, therapeutic proteins, antibodies and industrial enzymes. We developed a strategy of enzymatic deglycosylation of proteins in planta by co-expressing bacterial PNGase F (Peptide: N-glycosidase F) [2,3] Using this strategy, Pfs48/45 protein was produced in Nicotiana benthamiana in a non-N-glycosylated form and conformation-specific mAbs, produced against different epitops of native Pfs48/45 protein, recognized the deglycosylated form of Pfs48/45 2- to 6-fold better than they recognized the glycosylated form [2]. The lethal toxin neutralizing activity and immunogenicity of plant produced in vivo deglycosylated PA83 was much higher than those of in vitro deglycosylated PA83, or mutant forms, indicating potential differences in protein folding of in vivo, in vitro or mutant non-glycosylated forms [4]

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