Here, we present a protocol to produce an oral vaccine candidate against Type 1 diabetes in an edible plant.
Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required.
Since the plant molecular biology revolution in 1980s, plant-based systems for the production of biopharmaceuticals can be considered as an alternative to traditional systems based on microbial and mammalian cells1. Plants display several advantages over traditional platforms, with scalability, cost-effectiveness and safety being the most relevant2. The recombinant product can be purified from transformed plant tissue and then administered, either parenterally or orally and, moreover, transformed edible plant can be used directly for oral delivery. The oral route simultaneously promotes mucosal and systemic immunity, and it eliminates the need for needles and specialized medical personnel. Furthermore, oral delivery eliminates the complex downstream processing, which normally accounts for 80% of the total manufacturing cost of a recombinant protein3. All those advantages can be translated into savings in production, supplies and labor reducing the costs of each dose, making the drug affordable to most of the global population.
Several strategies, both for stable transformation and transient expression, were developed for the production of recombinant proteins in plants. Among them, a high-yield deconstructed plant virus-based expression system (e.g., magnICON) provides superior performance leading high yields of recombinant proteins over relatively short timescales4. Many examples of transient expression using the plant virus-based expression system in Nicotiana benthamiana plants are reported, being the gold standard production host. However, this model plant is not regarded as an edible species due to the alkaloids and other toxic metabolites that are accumulated in its leaves.
In this work, we describe the comparison between two edible plant systems, red beet (Beta vulgaris cv Moulin Rouge) and spinach (Spinacea oleracea cv Industria), for the expression of two candidate forms of the 65 kDa isoform of glutamic acid decarboxylase (GAD65), carried out by the plant virus-based vectors5. GAD65 is a major autoantigen associated to Type 1 Diabetes (T1D) and it is currently under investigation in human clinical trials to prevent or delay T1D by inducing tolerance6. The production of GAD65 in plants has been extensively studied in model plant species as Nicotiana tabacum and N. benthamiana4,5,6,7. Here, we describe the use of edible plant species for the production of the molecule in tissues that can be meant for a direct oral delivery. From a technical point of view, we studied and selected the system for plant agroinfiltration and the edible plant platform for GAD65 production by evaluating different parameters: the recombinant protein expression levels, the residual microbial charge in plant tissue meant for oral delivery, the resistance of GAD65 to the gastric digestion, and the bioequivalence of the transformed plants with the wild type.
1. Red beet and spinach cultivation
2. Transient expression through the deconstructed plant virus-based technology
3. Recombinant protein expression analysis
4. Plant material processing
5. Gastric digestion simulation and cell integrity analysis
6. Bioburden assay
7. Metabolite extraction
8. Liquid chromatography mass spectrometry analysis and data processing
In this work, the workflow for the development of an oral vaccine in edible plant tissues is presented. The focus of this work is the expression of a target protein in an edible host plant species and the characterization of the potential oral vaccine.
The first step involved the evaluation of the suitability of the plant virus-based expression technology to produce recombinant proteins in edible plant systems. For this aim, we first use the eGFP as a model protein and we expressed it into two edible leafy plant systems: red beet and spinach. Plants were manually agroinfiltrated with suspensions of A. tumefaciens carrying eGFP recombinant expression vectors. The fluorescent protein expression was visualized by western blot analysis (Figure 1A,B,D,E) and quantified under UV light. Results showed that the red beet system is characterized by a higher eGFP expression, reaching 544.9 ± 10.9 µg/g of fresh leaf weight (FLW) at 9 dpi (Figure 1C), than the spinach, which maximum eGFP levels (113.4 ± 0.3 µg/g FLW) were measured at 11 dpi (Figure 1F). For these reasons red beet was selected as expression host for all subsequent experiments.
According to Chen et al.10, the eGFP transient expression was tested by a vacuum method for infiltration, which is more suitable for the large scale vaccine production. Different dilutions of the overnight A. tumefaciens culture, ranging from 10-1 to 10-3, and different concentrations of detergent (0.005-0.05%) have been tested by comparing the eGFP accumulation level at the maximum expression dpi (9 dpi). The results found that higher bacterial titers produced greater eGFP yields (10-1 ~ 0.35). However, no significant differences were found using different detergent concentrations (data not shown).
Then, plants were vacuum infiltrated with an A. tumefaciens suspension at 0.35 OD600 and 0.01% of detergent. Once the expression platform and the delivery system were established, the expression of two forms of GAD65, GAD65mut and a N-terminally truncated form (∆87GAD65mut), was compared at the day of maximum expression, 5 and 11 dpi, respectively, as previously established11. After the TSP extraction from agroinfiltrated leaves, all samples were analyzed by western blot and the recombinant protein was relatively quantified by a densitometry analysis (Figure 2). Results highlighted the 20-fold higher performance of the ∆87GAD65mut form over the GAD65mut. The truncated form was therefore selected as preferred oral vaccine candidate, accumulating as high as 201.4 ± 29.3 µg/g FLW in red beet leaves at 11 dpi.
Finally, parameters for the development of a potential oral vaccine were evaluated. The recombinant protein integrity after freeze-drying of vacuum infiltrated red beet leaves expressing ∆87GAD65mut was assessed in comparison with the untreated (only frozen) tissue, by western blot analysis. As shown in Figure 3A, the target protein demonstrated to be stable after the lyophilization process.
The simulation of gastric digestion was carried out on the freeze-dried material by adding porcine gastric enzyme pepsin to a final concentration of 1 mg/mL or at a ratio of 1:20 to TSP. Both digestive treatment conditions resulted in the recombinant protein degradation, as demonstrated by western blot analysis (Figure 3C,D,E, data reported only for the pepsin final concentration of 1 mg/mL). The absence of a specific signal in the pellet samples after pepsin digestion (lanes pepsin, p 1-3), suggested that after freeze-drying treatment, the plant cells lost their integrity, leading to the target protein degradation.
The evaluation of cell integrity showed that when dried plant tissue was resuspended in buffer with neutral pH (pH 7.4), ∆87GAD65mut was partially solubilized, suggesting that at least some cells were broken during leaf grinding and drying. The resuspension of dried plant tissue in acidic conditions, instead, led to the detection of the ∆87GAD65mut only in the insoluble fraction. This was probably due to the ∆87GAD65mut precipitation caused by the low pH, in addition to the protein content of unbroken cells11. These assays indicate that freeze drying could be selected as treatment for the preparation of the vaccine candidate.
Furthermore, the residual microbial charge in the agroinfiltrated red-beet leaves was evaluated.
The bioburden assay displayed that the treatments exploited for the candidate vaccine preparation eliminated the bacterial load11.
The metabolic bioequivalence of ∆87GAD65mut and control red beet plants was assessed by fingerprints of primary and secondary metabolites and polar lipids generated by LC-MS from nine red beet plants expressing ∆87GAD65mut, nine agroinfiltrated controls and nine wild-type controls. PCA statistic revealed a wild-type plant cluster separated from all the other samples. No significant differences were instead highlighted between the ∆87GAD65mut and infiltrated and negative control plants. Furthermore, no significant difference among the three groups of plants in terms of polar lipid profiles was identified11.
Figure 1: Comparison of eGFP expression levels in agroinfiltrated red beet and spinach leaves. Western blot analysis (A, D) and corresponding loading controls (RuBisCO large subunit) stained with Coomassie Brilliant Blue (B, E) of protein extracts from eGFP-expressing leaf samples collected during the time-course analysis from 4 to 14 days post infection (dpi). The eGFP content of each leaf protein extract was quantified by fluorescence measurement (C, F). The results from agroinfiltrated red-beet leaves samples are displayed in the left panel, where the agroinfiltrated spinach samples are shown in the right one. Equal amounts of protein extracts have been loaded, 3.5 µg for the western blot and 30 µg for the Coomassie staining. An anti-eGFP antibody has been used as a probe in the western blot analysis. Side numbers indicate molecular mass markers in kDa. p.c., positive control, 10 ng of commercial recombinant human GAD65; n.c., negative control, extract from leaves infiltrated solely with A. tumefaciens carrying the 5’- and integrase modules. Error bars represent the standard deviation from three independent experiments. This figure has been modified from Bertini et al.11. Please click here to view a larger version of this figure.
Figure 2: GAD65mut and Δ87GAD65mut expression levels in red beet leaves. Western blot analysis (A) and corresponding loading control (RuBisCo large subunit) stained with Coomassie Brilliant Blue (B) of protein extracts from three red beet leaves expressing Δ87GAD65mut (left) and GAD65mut (right). An anti-GAD antibody has been used as a probe in the western blot analysis (the lanes were loaded with 20 μL of extract for GAD65mut and 1 μL of extract for Δ87GAD65mut). In the Coomassie stained gel, the same volume of protein extracts (10 μL/lane) was loaded for GAD65mut and Δ87GAD65mut. Side numbers indicate molecular mass markers in kDa. p.c., positive control, 10 ng of commercial recombinant human GAD65; n.c., negative control, extract obtained from leaves infiltrated only with A. tumefaciens carrying the 5’- and integrase modules. (C) Using the western blot positive control as a reference for a densitometric analysis, the relative expression levels of the two protein forms are plotted. Error bars represent the standard deviation from three independent experiments. This figure has been modified from Bertini et al.11. Please click here to view a larger version of this figure.
Figure 3: Oral vaccine candidate evaluation. On the left side, analyses of protein stability after freeze-drying treatment (A, B). Western blot analysis (A) and corresponding gel stained with Coomassie Brilliant Blue (B) representing three independent extracts from leaves expressing Δ87GAD65mut. Harvested leaves were directly frozen (fresh) or lyophilized at -50 °C, 0.04 mbar for 72 h (freeze dried). Different tissue:buffer ratios were employed during the TSP extraction in order to consider the water loss due to dehydration. Equal volume of extracts were loaded, 0.25 and 10 µL, for western blot and Coomassie staining respectively. An anti-GAD antibody has been used for the western blot probing Side numbers indicate molecular mass markers in kDa. n.c., negative control, extract from leaves infiltrated solely with A. tumefaciens carrying the 5’- and integrase modules. On the right side, in vitro simulated gastric digestion of Δ87GAD65mut (C,D,E). Western blot analyses (C, D) and corresponding gel stained with Coomassie Brilliant Blue (E) representing three independent extracts of leaves expressing Δ87GAD65mut after simulated gastric digestion. 1 mg/mL of pepsin has been added to 100 mg of lyophilized tissue, while a control sample without enzyme has been used. 24 µL and 16 µL, for supernatants (s) and pellets (p) respectively obtained in the final centrifugation step, have been used for SDS-PAGE analysis. Anti-GAD (C) and anti-LHCB2 (D) antibodies were used as probes in the western blot analysis. Side numbers indicate molecular mass markers in kDa. Please click here to view a larger version of this figure.
Primary metabolites (100 min) | Time (min) | % A | % B | Duration (min) | Type |
Initial | 0 | 100 | – | Initial condition | |
0 | 0 | 100 | 10 | Isocratic | |
10 | 15 | 85 | 15 | Gradient | |
25 | 15 | 85 | 5 | Isocratic | |
30 | 50 | 50 | 10 | Gradient | |
40 | 50 | 50 | 30 | Isocratic | |
70 | 0 | 100 | 1 | Gradient | |
71 | 0 | 100 | 29 | Isocratic (re-equilibrium) | |
Secondary metabolites (60 min) | Time (min) | % A | % B | Duration (min) | Type |
Initial | 98 | 2 | – | Initial condition | |
0 | 90 | 10 | 2 | Gradient | |
2 | 80 | 20 | 10 | Gradient | |
12 | 75 | 25 | 2 | Gradient | |
14 | 30 | 70 | 7 | Gradient | |
21 | 30 | 70 | 5 | Isocratic | |
26 | 10 | 90 | 1 | Gradient | |
27 | 10 | 90 | 14 | Isocratic | |
41 | 98 | 2 | 1 | Gradient | |
42 | 98 | 2 | 18 | Isocratic (re-equilibrium) | |
Polar lipids (90 min) | Time (min) | % A | % B | Duration (min) | Type |
Initial | 50 | 50 | – | Initial condition | |
0 | 0 | 100 | 10 | Gradient | |
10 | 0 | 100 | 65 | Isocratic | |
75 | 50 | 50 | 1 | Gradient | |
76 | 50 | 50 | 14 | Isocratic (re-equilibrium) |
Table 1: Gradient conditions for metabolite elution in LC-MS analysis.
Mass spectrometer components | Function | Parameters | ||||
Primary metabolites | Secondary metabolites | Polar lipids | ||||
Electrospray Ionization (ESI) Source | Nebulizing gas | 50 psi, 350 °C | 50 psi, 350 °C | - | ||
Drying gas | 10 L min-1 | 10 L min-1 | ||||
Atmospheric Pressure Chemical Ionization (APCI) Source | Nebulizing gas | - | - | 50 psi, 350 °C | ||
Drying gas | 10 L min-1 | |||||
Vaporizer | 450 °C | |||||
Ion trap and detector scan | Full scan mode | 13,000 m/z per second | 13,000 m/z per second | 13,000 m/z per second | ||
50-1,500 m/z | 50-1,500 m/z | 50-1,500 m/z | ||||
Scan range | 200 m/z | 400 m/z | 700 m/z | |||
Target mass | ||||||
Collision gas | Helium | |||||
Vacuum pressure | 1.4 x 10-5 mbar | |||||
Capillary source | +4,500 V | +4,500 V | +4,000 V | |||
End plate offset | -500 V | -500 V | -500 V | |||
Skimmer | 40 V | -40 V | 40 V | |||
Cap exit | 106 V | -121 V | 143.5 V | |||
Oct 1 DC | 12 V | -12 V | 12 V | |||
Oct 2 DC | 1.7 V | -1.7 V | 2 V | |||
Lens 1 | -5 V | 5 V | -5 V | |||
Lens 2 | -60 V | 60 V | -60 V | |||
ICC for positive ionization mode | 20 | |||||
ICC for negative ionization mode | 7 |
Table 2: Parameters for mass spectra acquisition in alternate positive and negative ionization modes.
In this study we showed preliminary analysis for the design of a candidate oral vaccine for autoimmune diabetes. The target protein for this experiment was a mutated form of the human 65 kDa Glutamate Decarboxylase, which production and functionality are easily detectable and measurable12. Its expression in different edible plant tissues was mediated by the vectors5, which mediate a high level of recombinant protein production in a very short time frame. The selection of the best candidate plant edible host was performed based on the eGFP expression in red beet and spinach leaves by manual agroinfiltration. This step could be critical for industrial scale-up because of the time-consuming procedure of manual agroinfiltration and the hardness in spinach leaf tissue infiltration.
The identification of the specific dpi of harvesting that gives the highest recombinant protein accumulation level for a recombinant protein is a critical step and need to be tested and selected on a case-by-case basis. The analysis of the fluorescent protein expression at different dpi (from 4 to 12), allowed to identify the day of maximum expression in each plant system. The comparison between the eGFP concentration (µg/g FLW), in these maximum expression days, highlighted that red beet is the best performing platform in terms of recombinant protein yields.
For this reason, red beet was further tested for the delivery of plant virus-based vectors by a vacuum system, which is considered more suitable for vaccine industrial large-scale production13.
Given that the standard vacuum infiltration protocol is optimized for tobacco species5, the set-up of a range of parameters for the procedure adjustment to the plant species considered is a critical point. Then, the red beet vacuum agroinfiltration protocol was optimized. Our evidence showed that the Agrobacterium dilution is crucial to improve the protein expression levels, while the detergent concentration to enhance the leaf permeability. The results showed that the plant system and the technology fit with this work purpose.
Two different forms of GAD65, GAD65mut and ∆87GAD65mut, that were previously characterized for their expression in N. benthamiana7, displaying different sub-cellular localization, were expressed in red beet by vacuum infiltration. Three biological replicates were prepared for every sample. Each biological replicate comprises a pool of three infiltrated leaves from different plants, sampled from 2 to 14 dpi for both the GAD65 forms. Their expression level was compared to select the highest accumulating protein in plant tissues.
The relative quantification by densitometry analysis demonstrated that the ∆87GAD65mut has a 20-fold higher expression level than its intact counterpart. This could be due to its cytosolic localization whilst GAD65mut, due to its residues that anchor this form to the cell membrane, has a lower yield7, reflecting a lower protein stability. ∆87GAD65mut average accumulation level in red beet leaf tissue was 201.4 ± 29.3 µg/g FLW, which is sufficient to start with the T1D oral vaccine development.
Finally, after the selection of the most suitable platform, technology and protein form, we proceeded by setting up a post-harvest treatment of the infected leaves. Since leaf tissue is composed of 95% of water, a dehydration treatment is useful to prevent microorganism contamination. Our results demonstrated that a freeze-drying treatment could be applied to the sample, without affecting the recombinant protein levels in the leaves. The 10-fold water removal by lyophilization eliminates the bacterial contamination (bioburden), including the recombinant A. tumefaciens used for the agroinfiltration procedure.
Furthermore, the analysis of protein bio encapsulation showed that the ∆87GAD65mut is completely digested following a simulated gastric digestion. This suggests that the freeze-drying treatment damages the plant cell wall, exposing the recombinant protein to the acidic and enzymatic composition of the gastric environment.
The maintenance of the protein or peptide molecule integrity over the gastrointestinal tract transition to the site of absorption represents an issue for oral drug delivery14. Hence, after dehydration treatment, the potential vaccine should be correctly formulated to overcome the gastric environment without losing its integrity. In the manufacture of the plant-derived GAD vaccine, the encapsulation in a relatively stable shell could be applied as a system to improve its efficiency allowing it to be protected and stable along the oral delivery route15.
Various technologies have been explored to overcome the problems associated with the oral delivery of macromolecules such as some recent studies on complexation of synthetic hydrogel with insulin which showed a high encapsulation efficiency and rapid insulin release in the intestine in a pH-dependent manner16,17.
Both the primary and secondary metabolite bioequivalence of plants expressing ∆87GAD65mut in comparison to infiltrated and non-infiltrated controls was investigated using PCA statistics. The differences between the infiltrated plants expressing ∆87GAD65mut and the infiltrated controls were not significant, whereas the non-infiltrated wild-type plants formed a separate cluster. These results suggest that the infiltrated plants are distinguished from untreated plants by the LC-MS analysis thanks to bacterial metabolites or metabolites produced by plants because of the infiltration, as already shown in literature13. Overall this analysis demonstrated that the accumulation of ∆87GAD65mut has little impact on the overall metabolism.
Altogether these results suggest that the potential oral vaccine, represented by freeze-dried red beet leaf tissue expressing ∆87GAD65mut obtained exploiting the plant virus-based expression technology, is suitable for experimental T1D oral immunotherapy trials. The plant virus expression vector technology has become very attractive in the transient expression field, due to its ability to produce foreign proteins both rapidly and at high levels. This technology is based on a deconstructed vector that carries only the RNA polymerase RNA dependent and movement protein5 and therefore it loses its infectivity in plants; as a consequence, its potential transmission to humans should be excluded.
The experimental protocol reported here could be extended to many different edible species, such as lettuce, Chenopodium capitatum and Tetragonia expansa. Since the leaves of these species, including red beet and spinach, whose was previously detected as good expression plant systems5, can be used as uncooked food, the technology proposed here might be used for manufacturing edible vaccines or for production of minimally processed functional food or feed.
The combination of recombinant protein/plant species, dpi of harvesting that gives the highest recombinant protein accumulation level need still to be determined empirically on a case by-case basis depending on the recombinant protein expressed and on the host plant species18.
The application of this technology for the production of oral vaccine hold great potential to become an alternative to conventional vaccines in the near future, in addition to combat oncolytic viruses, autologous vaccines for lymphomas and solid tumors, and monoclonal antibodies to target cancers19,20.
The authors have nothing to disclose.
This work was supported by the Joint Project “The use of plants for the production of an autoimmune diabetes edible vaccine (eDIVA)” (Project ID: 891854) funded by the University of Verona in the framework of the call 2014.
0.2-μm Minisart RC4 membrane filters | Sartorius-Stedim | 17764 | |
2–mercaptoethanol | Sigma | M3148 | Toxic; 4 % to make loading buffer with glycerol, SDS and Tris-HCl |
4-Morpholineethanesulfonic acid (MES) | Sigma | M8250 | pH 5.5 |
96-well plate | Sarstedt | 833924 | |
Acetic acid | Sigma | 27221 | Corrosive |
Acetonitrile LC-MS grade | Sigma | 34967 | |
Acetosyringone | Sigma | D134406 | Toxic – 0.1 M stock in DMSO |
Agar Bacteriological Grade | Applichem | A0949 | 15 g/L to make LB medium (pH 7.5 with NaOH) with Yeast extract, NaCl and Tryptone |
Ammonium formate | Sigma | 70221 | |
Anti-eGFP antibody | ABCam | ab290 | |
Anti-GAD 65/67 antibody | Sigma | G5163 | |
Anti-LHCB2 antibody | Agrisera | AS01 003 | |
Brilliant Blue R-250 | Sigma | B7920 | |
C18 Column | Grace | – | Alltima HP C18 (150 mm x 2.1 mm; 3 μm) Column |
C18 Guard Column | Grace | – | Alltima HP C18 (7.5 mm x 2.1 mm; 5 μm) Guard Column |
CalMag Grower | Peter Excel | 15-5-15 | Fertilizer |
Carbenicillin disodium | Duchefa Biochemie | C0109 | Toxic |
Chemiluminescence imaging system | BioRad | 1708370 | ChemiDoc Touch Imaging System |
Chloroform | Sigma | C2432 | |
Detergent | Sigma | P5927 | Polysorbate 20 |
Fluorescence reader | Perkin-Elmer | 1420-011 | VICTOR Multilabel Counter |
Formic acid LC-MS grade | Sigma | 94318 | |
Glycerol | Sigma | G5516 | 15 % to make loading buffer with Tris-HCl, SDS and 2–mercaptoethanol |
GoTaq G2 polymerase | Promega | M7841 | |
HCl | Sigma | H1758 | Corrosive |
HILIC Column | Grace | – | Ascentis Express HILIC (150 mm x 2.1 mm; particles size 2.7 μm) Column |
HILIC Guard Column | Grace | – | Vision HT HILIC (7.5 mm x 2.1 mm; 3 μm) Guard Column |
Horseradish peroxidase (HRP)-conjugate anti-rabbit antibody | Sigma | A6154 | Do not freeze/thaw too many times |
HPLC Autosampler | Beckman Coulter | – | System Gold 508 Autosampler |
HPLC System | Beckman Coulter | – | System Gold 128 Solvent Module HPLC |
Isopropanol | Sigma | 24137 | Flamable |
Kanamycin sulfate | Sigma | K4000 | Toxic |
KCl | Sigma | P9541 | 2 g/L with NaCl , Na2HPO4 and KH2PO4 to make PBS |
KH2PO4 | Sigma | P9791 | 2.4 g/L with NaCl , Na2HPO4 and KCl to make PBS |
Loading Buffer | |||
Luminol solution | Ge Healthcare | RPN2232 | Prepare the solution using the ECL Prime Western Blotting System commercial kit |
Lyophilizator | 5Pascal | LIO5P0000DGT | |
Mass Spectometer | Bruker Daltonics | – | Bruker Esquire 6000; the mass spectrometer was equipped with an ESI source and the analyzer was an ion trap |
Methanol | Sigma | 32213 | |
MgSO4 | Sigma | M7506 | |
Milk-blocking solution | Ristora | – | 3 % in PBS |
Na2HPO4 | Sigma | S7907 | Use with NaH2PO4 to make Sodium Phospate buffer |
NaCl | Sigma | S3014 | 80 g/L with KCl, Na2HPO4 and KH2PO4 to make PBS; 10 g/L to make LB medium (pH 7.5 with NaOH) with Yeast extract, Tryptone and Agar Bacteriological Grade |
NaH2PO4 | Sigma | S8282 | Use with Na2HPO4 to make Sodium Phospate buffer; 14.4 g/L to make PBS |
NaOH | Sigma | S8045 | |
Nitrocellulase membrane | Ge Healthcare | 10600002 | |
Pepsin from porcine gastric mucosa | Sigma | P7000 | |
Peroxidase substrate ECL | GE Healthcare | RPN2235 | Light sensitive material |
Pump Vacuum Press | VWR | 111400000098 | |
Reagent A | Sigma | B9643 | Use 50 parts of this reagent with 1 part of reagent B to prepare BCA working solution |
Reagent B | Sigma | B9643 | Use 1 part of this reagent with 50 parts of reagent A to prepare BCA working solution |
Rifampicin | Duchefa Biochemie | R0146 | Toxic – 25 mg/mL stock in DMSO |
SDS (Sodium dodecyl sulphate) | Sigma | L3771 | Flamable, toxic, corrosive-10 % stock; 3 % to make loading buffer with Tris-HCl, Glycerol and 2–mercaptoethanol |
Sodium metabisulphite | Sigma | 7681-57-4 | |
Sonicator system | Soltec | 090.003.0003 | Sonica® 2200 MH; frequency 40 khz |
Syringe | Terumo | – | |
Transparent fixed 300-µL insert glass tubes | Thermo Scientific | 11573680 | |
Trizma Base | Sigma | T1503 | Adjust pH with 1N HCl to make Tris-HCl buffer, use 1,5M Tris-HCl (pH 6.8) to make loading buffer with SDS, Glycerol and 2–mercaptoethanol |
Tryptone | Formedium | TRP03 | 10 g/L to make LB medium (pH 7.5 with NaOH) with Yeast extract, NaCl and Agar Bacteriological Grade |
Vacuum concentrator | Heto | 3878 F1-3 | Speed-vac System |
Water LC-MS grade | Sigma | 39253 | |
Yeast extract | Sigma | Y1333 | 5 g/L to make LB medium (pH 7.5 with NaOH) with Tryptone, NaCl and Agar Bacteriological Grade |