Une analyse comparative des recombinant expression des protéines dans différents bio-usines: bactéries, cellules d'insectes et Plant Systems
Summary
In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.
Abstract
Systèmes à base de plantes sont considérées comme une plate-forme précieuse pour la production de protéines recombinantes en raison de leur potentiel bien documenté pour la production à faible coût flexible de haute qualité, produits bioactifs.
Dans cette étude, nous avons comparé l'expression d'une protéine cible recombinante humaine dans les cultures traditionnelles à base de fermentation de cellules (bactériennes et d'insectes) avec les systèmes d'expression à base de plantes, à la fois transitoires et stables.
Pour chaque plate-forme, nous avons décrit la mise en place, l'optimisation et la longueur du processus de production, la qualité du produit final et les rendements et nous avons évalué les coûts de production provisoires, spécifiques de la protéine recombinante cible sélectionnée.
Dans l'ensemble, nos résultats indiquent que les bactéries ne sont pas appropriés pour la production de la protéine cible en raison de son accumulation dans les corps d'inclusion insolubles. D'autre part, les systèmes à base de plantes sont des plateformes polyvalentes tchapeau permet la production de la protéine choisie à plus faibles que les coûts-baculovirus / système de cellule d'insecte. En particulier, les lignées transgéniques stables affichent le plus haut rendement du produit final et transitoires plantes exprimant le processus de développement plus rapide. Cependant, toutes les protéines recombinantes peuvent bénéficier de systèmes à base de plantes, mais la meilleure plate-forme de production devraient être déterminées empiriquement avec une approche au cas par cas, comme décrit ici.
Introduction
Recombinant proteins are commercially mass-produced in heterologous expression systems with the aid of emerging biotechnology tools. Key factors that have to be considered when choosing the heterologous expression system include: protein quality, functionality, process speed, yield and cost.
In the recombinant protein field, the market for pharmaceuticals is expanding rapidly and, consequently, most biopharmaceuticals produced today are recombinant. Proteins can be expressed in cell cultures of bacteria, yeasts, molds, mammals, plants and insects, as well as in plant systems (either via stable- or transient-transformation) and transgenic animals; each expression system has its inherent advantages and limitations and for each target recombinant protein the optimal production system has to be carefully evaluated.
Plant-based platforms are arising as an important alternative to traditional fermenter-based systems for safe and cost-effective recombinant protein production. Although downstream processing costs are comparable to those of microbial and mammalian cells, the lower up-front investment required for commercial production in plants and the potential economy of scale, provided by cultivation over large areas, are key advantages.
We evaluated plants as bioreactors for the expression of the 65 kDa isoform of human glutamic acid decarboxylase (hGAD65), one of the major autoantigen in Type 1 autoimmune diabetes (T1D). hGAD65 is largely adopted as a marker, both for classifying and monitoring the progression of the disease and its role in T1D prevention is currently under investigation in clinical trials. If these trials are successful, the global demand for recombinant hGAD65 will increase dramatically.
Here, we focus on the expression of the enzymatically inactive counterpart of hGAD65, hGAD65mut, a mutant generated by substituting the lysine residue that binds the cofactor PLP (pyridoxal-5′-phosphate) with an arginine residue (K396R)1.
hGAD65mut retains its immunogenicity and, in plant and insect cells, accumulates up to ten-fold higher than hGAD65, its wild-type counterpart. It was hypothesized that the enzymatic activity of hGAD65 interferes with plant cell metabolism to such an extent that it suppresses its own synthesis, whereas hGAD65mut, the enzymatically-inactive form, can be accumulated in plant cells to higher levels.
For the expression of hGAD65mut, the use of different technologies, widely used in plant biotechnology, was explored here and compared to traditional expression platforms (Escherichia coli and Baculovirus/insect cell-based).
In this work, the recombinant platforms developed for the expression of hGAD65mut comprising traditional and plant-based systems were reviewed and compared on the basis of process speed and yield, and of final product quality and functionality.
Protocol
Representative Results
Discussion
Dans cette étude, trois modes différents ont été comparés pour l'expression d'une protéine recombinante humaine: des cellules bactériennes, des cellules de baculovirus / insectes et de plantes. La plate-forme à base de plantes a été étudiée plus en exploitant trois technologies d'expression largement utilisés (c.-à-transitoire – MagnICON et pK7WG2 base – et stables). La protéine cible choisie pour cette expérience, hGAD65mut, a déjà été exprimé dans différents systèmes 13…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the COST action ‘Molecular pharming: Plants as a production platform for high-value proteins’ FA0804. The Authors thank Dr Anatoli Giritch and Prof. Yuri Gleba for providing the MagnICON vectors for research purposes.
Materials
| Yeast extract | Sigma | Y1333 | |
| Tryptone | Formedium | TRP03 | |
| Agar Bacteriological Grade | Applichem | A0949 | |
| Sf-900 II SFM medium | Gibco | 10902-088 | |
| Grace’s Insect Medium, unsupplemented | Gibco | 11595-030 | |
| Cellfectin II Reagent | Invitrogen | 10362-100 | |
| MS medium including vitamins | Duchefa Biochemie | M0222 | |
| Sucrose | Duchefa Biochemie | S0809 | |
| Plant agar | Duchefa Biochemie | P1001 | |
| Ampicillin sodium | Duchefa Biochemie | A0104 | Toxic |
| Gentamycin sulphate | Duchefa Biochemie | G0124 | Toxic |
| Ganciclovir | Invitrogen | I2562-023 | |
| Carbenicillin disodium | Duchefa Biochemie | C0109 | Toxic |
| Kanamycin sulfate | Sigma | K4000 | Toxic |
| Rifampicin | Duchefa Biochemie | R0146 | Toxic – 25 mg/ml stock in DMSO |
| Streptomycin sulfate | Duchefa Biochemie | S0148 | Toxic |
| Spectinomycin dihydrochloride | Duchefa Biochemie | S0188 | |
| IPTG (Isopropil-β-D-1-tiogalattopiranoside) | Sigma | I5502 | Toxic |
| MES hydrate | Sigma | M8250 | |
| MgCl2 | Biochemical | 436994U | |
| Acetosyringone | Sigma | D134406 | Toxic – 0.1 M stock in DMSO |
| Syringe (1 ml) | Terumo | ||
| MgSO4 | Fluka | 63136 | |
| BAP (6-Benzylaminopurine) | Sigma | B3408 | Toxic |
| NAA (Naphtalene acetic acid) | Duchefa Biochemie | N0903 | Irritant |
| Cefotaxime | Mylan generics | ||
| Trizma base | Sigma | T1503 | Adjust pH with 1 N HCl to make Tris-HCl buffer |
| HCl | Sigma | H1758 | Corrosive |
| NaCl | Sigma | S3014 | 1 M stock |
| KCl | Sigma | P9541 | |
| Na2HPO4 | Sigma | S7907 | |
| KH2PO4 | Sigma | P9791 | |
| PMSF (Phenylmethanesulfonylfluoride) | Sigma | P7626 | Corrosive, toxic |
| Urea | Sigma | U5378 | |
| β-mercaptoethanol | Sigma | M3148 | Toxic |
| Tween-20 | Sigma | P5927 | |
| Hepes | Sigma | H3375 | |
| DTT (Dithiothreitol) | Sigma | D0632 | Toxic – 1 M stock, store at -20 °C |
| CHAPS | Duchefa Biochemie | C1374 | Toxic |
| Plant protease inhibitor cocktail | Sigma | P9599 | Do not freeze/thaw too many times |
| SDS (Sodium dodecyl sulphate) | Sigma | L3771 | Flammable, toxic, corrosive – 10% stock |
| Glycerol | Sigma | G5516 | |
| Brilliant Blue R-250 | Sigma | B7920 | |
| Isopropanol | Sigma | 24137 | Flammable |
| Acetic acid | Sigma | 27221 | Corrosive |
| Anti-Glutamic acid decarboxylase 65/67 | Sigma | G5163 | Do not freeze/thaw too many times |
| Horseradish peroxidase (HRP)-conjugate anti-rabbit antibody | Sigma | A6154 | Do not freeze/thaw too many times |
| Sf9 Cells | Life Technologies | 11496 | |
| BL21 Competent E.coli | New England Biolabs | C2530H | |
| Protein A Sepharose | Sigma | P2545 | |
| Cell culture plates | Sigma | CLS3516 | |
| Radio Immuno Assay kit | Techno Genetics | 12650805 | Radioactive material |
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