박테리아, 곤충 세포 및 공장 시스템 : 재조합 단백질 다른 Biofactories에서 표현의 비교 분석
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
식물 – 기반 시스템은 높은 품질의 생체 활성 제품의가요 성, 저비용 생산을 위해 자신의 잘 문서화 전위의 결과로 재조합 단백질의 생산에 유용한 플랫폼 여겨진다.
본 연구에서 우리는 과도하고 안정적인 식물 기반 발현 시스템, 전통적인 발효기 계 세포 배양 (세균성 및 곤충)에서 표적 재조합 인간 단백질의 발현을 비교 하였다.
각 플랫폼을 위해, 우리는 셋업, 최적화 및 제조 공정의 길이, 최종 제품의 품질과 수율을 설명하고 우리는 선택된 대상 재조합 단백질에 특이 잠정 생산 비용을 평가 하였다.
전반적으로, 우리의 결과는 박테리아 인해 불용성 봉입체 내에서 축적 목적 단백질의 생산에 적합하지 않은 것을 나타낸다. 한편, 식물 기반 시스템은 다목적 플랫폼 t는모자 배큘로 바이러스 / 곤충 세포 시스템보다 낮은 비용으로 선택된 단백질의 생산을 허용한다. 특히, 안정된 형질 전환 라인은 최종 생성물의 높은 수율 및 과도 발현하는 식물 빠른 프로세스 개발을 표시. 그러나, 모든 재조합 단백질은 식물 기반 시스템의 혜택을 누릴 수 있지만, 여기에 설명 된대로 최고의 생산 플랫폼, 사례 별 접근 방식을 경험적으로 결정되어야한다.
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
박테리아 세포, 배큘로 바이러스 / 곤충 세포 및 식물 : 본 연구에서는 세 가지 플랫폼은 재조합 인간 단백질의 발현을 비교 하였다. (- MagnICON 및 pK7WG2 기반 – 안정적인 즉, 과도) 식물 기반 플랫폼은 더 세 널리 사용되는 표현 기술을 이용하여 탐구 하였다. 이 실험 hGAD65mut 위해 선택된 표적 단백질은 이전에 다른 시스템 (13)으로 표현되었고, 그것의 생산 및 기능은 쉽게 검출 및 ?…
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 |
References
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