細菌、昆虫細胞およびプラントシステムズ：異なるバイオファクトリーでの組換えタンパク質発現の比較分析
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
植物ベースのシステムは、高品質の、生理活性製品の柔軟な、低コスト生産のためのそれらの十分に立証された電位の結果として、組換えタンパク質の生産のための貴重なプラットフォームと考えられている。
本研究では、一過性で安定した植物に基づく発現系、伝統的な発酵槽に基づく細胞培養物（細菌および昆虫）で標的のヒト組換えタンパク質の発現を比較した。
各プラットフォームのために、我々は、セットアップ、最適化、製造工程の長さは、最終製品の品質及び収率を記載し、我々は、選択された標的組換えタンパク質に特異的な仮の製造コストを評価した。
全体として、我々の結果は、細菌は、不溶性の封入体中のその蓄積による標的タンパク質の生産に適していないことを示している。一方、植物ベースのシステムは、多目的プラットフォームトンである帽子は、バキュロウイルス/昆虫細胞系よりも低コストで選択されたタンパク質の産生を可能にする。具体的には、安定したトランスジェニック系統は、最終生成物の最高収率及び過渡発現する植物最速プロセス開発を示した。しかしながら、全ての組換えタンパク質は、植物ベースのシステムから利益を得ることができるが、ここで説明したように最高の生産プラットフォームは、ケースバイケースのアプローチを経験的に決定されるべきである。
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
細菌細胞、バキュロウイルス/昆虫細胞および植物：本研究では三つの異なるプラットフォームは、組換えヒトタンパク質の発現を比較した。植物ベースのプラットフォームは、さらに、（かつ安定した- -ベースMagnICONとpK7WG2 すなわち 、一過性の）3広く使用されている発現技術を利用して調査した。この実験で、hGAD65mutのために選択された標的タンパク質は、以前に別のシステム<…
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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