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.
Plant-baserede systemer betragtes som en værdifuld platform til produktion af rekombinante proteiner som følge af deres veldokumenterede muligheder for fleksibel og billig produktion af høj kvalitet, bioaktive produkter.
I denne undersøgelse sammenlignede vi ekspressionen af et target human rekombinant protein i traditionelle fermentorbetingelser baseret cellekulturer (bakterielle og insekter) med plante-baserede ekspressionssystemer, både forbigående og stabile.
For hver platform, vi beskrev opsætning, optimering og længden af fremstillingsprocessen, det endelige produkt, og udbytterne og vi vurderet foreløbige produktionsomkostninger, der er specifikke for det valgte mål rekombinant protein.
Samlet set vores resultater viser, at bakterier er uegnet til produktion af målproteinet grundet dens akkumulering i uopløselige inklusionslegemer. På den anden side, plante-baserede systemer er alsidige platforme that tillade produktionen af den valgte protein ved lavere omkostninger end Baculovirus / insektcelle-systemet. Især stabile transgene linier viste det højeste udbytte af slutproduktet og forbigående udtrykker planter den hurtigste procesudvikling. Dog kan ikke alle rekombinante proteiner gavn af plante-baserede systemer, men den bedste produktionsplatform skal bestemmes empirisk med en sag til sag-tilgang, som beskrevet her.
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.
I denne undersøgelse tre forskellige platforme blev sammenlignet for ekspression af et rekombinant humant protein: bakterieceller, Baculovirus / insektceller og planter. Anlægget-baseret platform blev yderligere udforsket ved at udnytte tre udbredte udtryk teknologier (dvs. forbigående – MagnICON og pK7WG2 baserede – og stabile). Målproteinet valgt til dette eksperiment, hGAD65mut, er tidligere blevet udtrykt i forskellige systemer 13 og produktionen og funktionalitet er let påviselige og målel…
The authors have nothing to disclose.
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.
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 |