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.
Växtbaserade system anses en värdefull plattform för produktion av rekombinanta proteiner som ett resultat av deras väldokumenterade potentialen för den flexibla, lågkostnadsproduktion av hög kvalitet, bioaktiva produkter.
I denna studie jämförde vi uttrycket av ett mål humant rekombinant protein i traditionella fermenteringsbaserade cellkulturer (bakterie- och insekts) med växtbaserade uttryckssystem, både övergående och stabila.
För varje plattform, beskrev vi set-up, optimering och längd av produktionsprocessen, den slutliga kvalitet och avkastningen och vi utvärderade preliminära produktionskostnader, som är specifika för det valda målet rekombinant protein.
Sammantaget indikerar våra resultat att bakterier är olämplig för framställning av målproteinet på grund av dess ackumulering inom olösliga inklusionskroppar. Å andra sidan, växtbaserade system är mångsidiga plattformar thatt tillåta produktionen av det valda proteinet vid lägre-kostnader än Baculovirus / insektscellsystemet. Särskilt stabila transgena linjer uppvisade den högsta utbytet av den slutliga produkten och övergående uttryckande växter den snabbaste processutveckling. Dock kan inte alla rekombinanta proteiner gynnas växtbaserade system, men det bästa produktionsplattform bör bestämmas empiriskt med en bedömning från fall till fall, som beskrivs här.
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 denna studie tre olika plattformar jämfördes för uttrycket av ett rekombinant humant protein: bakterieceller, baculovirus / insektsceller och växter. Anläggningen baserade plattformen utforskas ytterligare genom att utnyttja tre vanligt förekommande uttryck teknik (dvs, övergående – MagnICON och pK7WG2 baserade – och stabila). Målproteinet valdes för detta experiment, hGAD65mut, tidigare har uttryckt i olika system 13, och dess produktion och funktionalitet är lätt att upptäcka och mä…
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 |