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

Mamedov, T. G.; Yusibov, Vidadi

doi:10.4161/bioe.23449

Cited by 13 publications

(12 citation statements)

References 25 publications

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“…The good linear correlation between the chemical lysis and mechanical reference extractions will facilitate a comparison to earlier results and data from whole plant extracts, and should be verified with other proteins such as monoclonal antibodies (Sack et al, 2015), vaccine candidates (Jones et al, 2013), and enzymes (Mamedov and Yusibov, 2013). In order to improve the compatibility of the lysis buffer with the activity of different proteins and diverse analysis methods, it would be useful to screen for components to replace EDTA and/or SDS, such as non-ionic or zwitterionic detergents like Triton X-100 or CHAPS (Shehadul Islam et al, 2017).…”

Section: Chemical Extraction Releases 91% Of Secreted Dsred Relative mentioning

confidence: 76%

Robot Cookies – Plant Cell Packs as an Automated High-Throughput Screening Platform Based on Transient Expression

Gengenbach

Opdensteinen

Buyel

2020

Front. Bioeng. Biotechnol.

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The high-throughput screening of recombinant protein expression is advantageous during early process development because it allows the identification of optimal expression constructs and process conditions. Simple screening platforms based on microtiter plates are available for microbes and animal cells, but this was not possible for plants until the development of plant cell packs (PCPs), also known as "cookies," which provide a versatile and scalable screening tool for recombinant protein production. PCPs are prepared from plant cell suspension cultures by removing the medium and molding the biomass. PCPs can be cast into 96-well plates for highthroughput screening, but the manual handling effort currently limits the throughput to ∼500 samples per day. We have therefore integrated the PCP method with a fully automated laboratory liquid-handling station. The "robot cookies" can be prepared and infiltrated with Agrobacterium tumefaciens by centrifugation, minimizing operator handling and reducing the likelihood of errors during repeated runs, such as those required in a design of experiments approach. The accumulation of fluorescent protein in the cytosol, apoplast, endoplasmic reticulum or plastids is easily detected using an integrated plate reader, reducing the inter-experimental variation to <5%. We also developed a detergent-based chemical lysis method for protein extraction in a 96-well format, which was adapted for automated downstream processing using miniaturized columns allowing subsequent protein analysis. The new automated method reduces the costs of the platform to <0.5 € per PCP infiltration (a saving of >50%) and facilitates a five-fold increase in throughput to >2500 samples per day.

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Section: Chemical Extraction Releases 91% Of Secreted Dsred Relative mentioning

confidence: 76%

Robot Cookies – Plant Cell Packs as an Automated High-Throughput Screening Platform Based on Transient Expression

Gengenbach

Opdensteinen

Buyel

2020

Front. Bioeng. Biotechnol.

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“…We hypothesized that it could be due to aberrant N-glycosylation of the Pfs48/45 protein. To address this need, in our previous efforts, we developed a strategy for the production of proteins in a non-glycosylated form, by co-expression of target proteins with the bacterial deglycosylation enzyme, PNGase F 11,30 . It was demonstrated that though removal of oligosaccharides from glycosylated proteins by PNGase F does cause an amino acid change in the produced deglycosylated protein, the strategy significantly improved the protein quality 11,24,29 .…”

Section: Discussionmentioning

confidence: 99%

“…There were attempts to produce a full-length Pfs48/45 protein in N . benthamiana plant using a transient expression system, but the resulting plant produced antigen showed very low TB activity 11,30 . It was hypothesized that the low TB activity may be associated with aberrant N-glycosylation of Pfs48/45 in plant system.…”

Section: Introductionmentioning

confidence: 99%

A Plant-Produced in vivo deglycosylated full-length Pfs48/45 as a Transmission-Blocking Vaccine Candidate against malaria

Mamedov

Cicek

Miura

et al. 2019

Sci Rep

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Pfs48/45 is a leading antigen candidate for a transmission blocking (TB) vaccine. However, efforts to produce affordable, safe and correctly folded full-length Pfs48/45 using different protein expression systems have not produced an antigen with satisfactory TB activity. Pfs48/45 has 16 cysteines involved in disulfide bond formation, and the correct formation is critical for proper folding and induction of TB antibodies. Moreover, Pfs48⁄45 is not a glycoprotein in the native hosts, but contains potential glycosylation sites, which are aberrantly glycosylated during expression in eukaryotic systems. Here, we demonstrate for the first time that full length, Endo H in vivo enzymatic deglycosylated Pfs48/45 antigen is produced at a high level in plants and is structurally stable at elevated temperatures. Sera from mice immunized with this antigen showed strong inhibition in SMFA. Thus, Endo H in vivo enzymatic deglycosylated Pfs48/45 is a promising candidate for the development of an affordable TB vaccine, which may have the potential to save millions.

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“…and biological activity. Recently, 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 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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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.

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

Cited by 13 publications

References 25 publications

Robot Cookies – Plant Cell Packs as an Automated High-Throughput Screening Platform Based on Transient Expression

Robot Cookies – Plant Cell Packs as an Automated High-Throughput Screening Platform Based on Transient Expression

A Plant-Produced in vivo deglycosylated full-length Pfs48/45 as a Transmission-Blocking Vaccine Candidate against malaria

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

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