Project protocol — Contents
Workflow and sampling
Equipment
Reagents, supplies, and solutions
Procedures
Definitions and calculations
Data
References
Workflow
• Cell culture incubator heated at 37°C with continuous shaker
• Spectrophotometer for cell density measurement
• Sorvall centrifuge with Sorvall SS-34 rotor and refrigeration down to 4°C temperature
• French press (Spectronic Instruments) for lysing cells up to 1000 psi
• Hisâ¢Bind column (Novagen)
• PD-10 column (Pharmacia)
• Anion-exchange chromatography (MonoQ and Q sepharose columns on the fast protein liquid chromatography system; Pharmacia)
• SDS poly-acrylamide gel electrophoresis (PAGE) system
• Gel drying kit (Promega)
Reagents, supplies, solutions Â
• pET cloning and expression system for recombinant proteins
• 150 mm culture plates
• LB/carbenicillin (50 µg/mL) culture plat
• phosphate buffered saline (PBS)
• Neutral Red Solution (Sigma N-2889) diluted 1:8 in bone marrow media (BMM)
• 0.45-µM syringe tip filter
• 30000 NMWL Centricon Plus-20 centrifugal filter (Millipore)
• Minitan Ultrafiltration System (Millipore)
• Centri-Prep concentrator (Millipore)
• 10% Tris-glycine SDSâPAGE gels with a 4% stacking gel (Bio-Rad)
• 96-well tissue culture plate (Costar)
• Neutral Red stain (Sigma N 2889)Tissue culture and media
• J774 macrophage cell line: anthrax-susceptible control (BALB/c derived) (gift from Dr Michael Starnbach, Harvard Medical School)
• IC-21 macrophage cell line: anthrax-resistant control (C57BL/6 derived) (American Type Culture Collection, Waldorf, MD, USA)
• Basic media: RPMI-1640 (Life Technologies) containing 10% heat-inactivated fetal bovine serum (Life Technologies), 200 μM L-glutamine (Life Technologies), 10 μg/mL streptomycin, and 10 units/mL penicillin (Life Technologies)
• Mouse L-cell conditioned media: as a source of granulocyte/macrophage colony-stimulating factor. A 175-cm2 tissue culture flask (Falcon Scientific Plastics) was seeded with 2.5 Ã 105 L-cells in 50 mL of basic medium and incubated just until confluency was achieved. The supernatant medium was then filtered through a 0.2-µm filter and frozen in 30 or 50 mL aliquots at â80°C.
• Bone marrow media (BMM): RPMI 1640 containing 20% heat-inactivated fetal bovine serum, 30% L-cell -conditioned media, 200 µM L-glutamine, 10 µg/mL streptomycin and 10 units/mL penicillin
• BMM/PA: BMM containing 1 μg/mL recombinant anthrax Protein Antigen (rPA)
Miscellaneous solutions
• Resuspension buffer: 50 mM Tris-HCl (pH 8.0), 2 mM EDTA containing 2 mM phenylmethylsulphonyl fluoride (PMSF; Sigma P 7626), 2 μg/mL pepstatin (Sigma P 4265), 2 μg/mL leupeptin (Sigma L 2884), 1% v/v aprotinin (Sigma A 6279) and 2 μg/mL antipain (Sigma A 0914).
• Binding buffer: Hisâ¢Bind buffer kit (Novagen) with 2 mM PMSF (Sigma P 7626), 2 μg/mL pepstatin (Sigma P 4265), 2 μg/mL leupeptin (Sigma L 2884), 1% v/v aprotinin (Sigma A 6279) and 2 μg/mL antipain (Sigma A 0914)
• Elution buffer: containing 373 mM imidazole, 500 mM NaCl and 20 mM Tris-HCl, pH 7.9
• 6 Ã SDS sample buffer: containing 270 mM Tris-HCl (pH 6.8), 10% SDS, 30% glycerol, 0.0012% bromophenol blue, and 600 mM dithiothreitol (DTT)
Toxins
Submitting PI note: "...native LF prepared from B. anthracis and the recombinant LF purified from the E. coli expression clones behaved virtually identically, they are referred to interchangeably as LF."
Lethal factor (LF) is purified from B. anthracis Sterne strain cultures using a published protocol (Roberts et al, 1998). Recombinant lethal factor (rLF) and recombinant protective antigen (rPA) are prepared in E. coli, as described below (constructs provided by Dr John Collier, Harvard Medical School).
For toxin preparation, all centrifugation steps are performed at 5000 × g in a Sorvall SS-34 rotor at 4°C (unless noted otherwise). Likewise, all cell types are incubated at 37°C, 5% CO2 in a humidified atmosphere.
Recombinant lethal factor (rLF) purification method
A liter of starting culture can yield approximately 1 mg of purified LF protein.
Bacterial culture
1) rLF protein expression is induced using a modification of the pET cloning and expression system.
2) E. coli strain BL21(DE3)/pET15bLFwt (rLF) is struck out onto a LB/carbenicillin (50 µg/mL) culture plate and incubated overnight.
3) 10 mL of LB/carbenicillin (50 µg/mL) is inoculated with a single colony and incubated with continuous shaking for approximately 3 h to an optical density (OD600) of 0.6â0.8.
4) 100 mL of LB/carbenicillin (50 µg/mL) is inoculated with the above culture and then incubated for 3 h with continuous shaking to an OD600 of 0.6â0.8 and then stored overnight at 4°C.
5) Cultured bacterial cells are then harvested by centrifugation, resuspended in 5 mL of LB, used to inoculate 1 L of LB/carbenicillin (50 µg/mL), and then incubated with continuous shaking for 2.5 h to an OD600 of 0.6â0.8.
6) Protein expression is induced by making the culture in 1 mM in IPTG, 500 μM in ZnSO4, 500 μM in CaCl2, 500 μM in MgSO4 and incubating overnight at 15°C with continuous shaking.
7) The culture is cooled to 4°C by incubating on ice for approximately 30 min. Bacterial cells are harvested by centrifugation and then resuspended in 250 mL of ice-cold 50 mM Tris, pH 8.0, 2 mM EDTA solution.
8) Bacterial cells are then harvested by centrifugation, the supernatant discarded, and the resulting cell pellets stored at â80°C until needed.Bacterial cell lyses and protein derivation
1) Stored frozen cell pellets are thawed for 10 min on ice, resuspended in 40 mL of ice-cold resuspension buffer, harvested by centrifugation, and then resuspended in 40 mL of ice-cold binding buffer.
2) The resuspended cells are lysed using a French press with a pressure of 1000 psi.
3) Cell lysate from the French press is cleared by centrifugation and supernatant is filtered through a 0.45 µM syringe tip filter.
4) Filtrate is immediately loaded onto the Hisâ¢Bind column at 4°C and processed according to the manufacturer's instructions using an ice-cold elution buffer.
5) Five 5 mL fractions of elute are collected, and then 25 µL samples of each are run on an SDSâPAGE gel to identify the fractions containing bacterial protein of the expected molecular weight (83 kDa).
Bacterial protein purification
1) Following manufacturer's instructions, 5 mL fractions containing rLF are combined and desalted using an anion-exchange chromatography (PD-10) column, equilibrated with 20 mM Tris and pH 8.0.
2) Desalted samples are then concentrated using a 30000 NMWL Centricon Plus-20 centrifugal filter according to the manufacturer's instructions.
3) The sample is then further purified using anion-exchange chromatography (MonoQ column on the fast protein liquid chromatography system) using a 20 mM Tris-HCl, pH 8.0, buffer and a linear gradient of 100â400 mM NaCl over a period of 40 min.
4) Fractions (1 mL) containing rLF are identified by SDSâPAGE, stored at â80°C, and eluted at approximately 250 mM NaCl.
5) The protein is purified to > 90% homogeneity, and potency, as measured in cytotoxicity assays, is consistent with that exhibited by native LF.
Protective antigen (PA) purification methods
A liter of starting culture can yield approximately 0.4 mg of purified protein PABacterial culture and derivation of periplasmic extract
1) Two liters of the E. coli strain BL21(DE3)/pET22b-PAwt (rPA) are grown, incubated with continuous shaking, to an OD600 of 0.6â0.8; culture is primed for induction at 30°C incubation for 3 h with the addition of 1 mM IPTG; induced cells are harvested by centrifugation for 10 min at 2000 Ã g in a Sorvall SS-34 rotor at 4°C.
2) The cell pellet is resuspended in 800 mL solution with 30 mM Tris, pH 8.0, 20% sucrose to derive the periplasmic extract.
3) After making the suspension in 1 mM in EDTA, pH 8.0, the cells are incubated for an additional 10 min.
4) The cells are spun down by centrifugation for 10 min, the pellet is resuspended in 800 mL of ice-cold 5 mM MgSO4 and incubated on ice for 10 min.
5) The resulting periplasmic extract is cleared by centrifugation for 10 min, and the pellet generated is discarded while the supernatant is used for the subsequent purification step.
6) The periplasmic extract is concentrated to a volume of 50 mL using the Minitan Ultrafiltration System.
7) The concentrated periplasmic extract is then diluted to 500 mL with 20 mM Tris, pH 8.0, and reconcentrated to 50 mL volume using the Minitan Ultrafiltration System.
Protective antigen purification
1) Generated protein suspension above is further purified by anion-exchange chromatography (Q sepharose column) with 20 mM Tris-HCl, pH 8.0, and a linear gradient of 100â450 mM NaCl over a period of 50 min.
2) rPA is then eluted using 250â350 mM NaCl solution.
3) Fractions (2 mL) containing rPA (which has a molecular weight of 83 kDa) are identified by SDSâPAGE, pooled and concentrated using a Centri-Prep concentrator according to the manufacturer's instructions.
4) The resulting 2 mL fractionated solution is further purified by anion-exchange chromatography (Mono Q column on the fast protein liquid chromatography system) with 20 mM Tris-HCl, pH 8.0, and a linear gradient of 50â550 mM NaCl over a period of 110 min.
5) The rPA protein is eluted with approximately 300 mM NaCl solution.
6) Using SDSâPAGE method, fractions containing rPA are identified.
7) The protein is further purified to > 90% homogeneity, and the potency, as measured in cytotoxicity assays, is comparable with that exhibited by other rPA.
I. Tissue culture
All cell types are incubated at 37°C, 5% CO2 in a humidified atmosphere.
a. Primary macrophages: bone marrow is flushed from mouse femurs to obtain approximately 4 × 106 cells and cultured in bone marrow media (BMM) for 6-7 d at 37°C in 100 mm polystyrene petri dishes, allowing weak adherence of macrophages and easy harvesting by scraping, to obtain mature, differentiated macrophages.
b. J774 macrophages (anthrax-susceptible control): 2 × 106 cells are seeded in a 100 mm polystyrene Petri dish with 30 mL of basic medium; passed 2x/wk by harvesting cells using a cell scraper, removing old media by centrifugation at 228 × g for 10 min, and resuspending cells in fresh basic medium and seeding 1 × 105 cells into a new 100 mm polystyrene Petri dish with 30 mL of basic medium.
c. IC-21 macrophages (anthrax-resistant control): 2 × 106 cells are seeded in a 100 mm polystyrene Petri dish with 25 mL of basic medium; cultures are fed 3x/wk with 5 mL of basic medium; upon reaching confluency, cells are subcultured by rinsing with Ca–Mg-free PBS, incubating in 10–15 mL of PBS for 10–15 min and dislodging cells by pipetting the PBS up and down; cell suspension is then transferred into a new Petri dish containing fresh basic medium at a 1:2 to 1:4 ratio.II. Anthrax toxin assay
a. Lethal factor (10 µg/mL) is added to BMM/PA. Serial dilutions are made to yield four concentrations of LF (1x105 ng/mL to 10 ng/mL). BMM/PA without LF is used as a control.
b. In a 96-well plate, 10 µL of the control (BMM/PA) and each dilution (BMM/PA with LF) are aliquoted and stored at 4°C.
c. Macrophages are rinsed twice with 10 mL of BMM, then 50 mL of fresh media is added.
d. Macrophages are harvested by scraping 1/3 of the cultured cell plate surface, and then transferring them into a 5 mL tube.
e. Macrophage cells are counted and resuspended, such that 4.5 X 104 cells per 90 µL BMM/PA are added to each well.
f. Cells are gently mixed by tapping the plate several times and incubated 4 h to overnight.
g. Cells are stained by making the media 1% Neutral Red.
h. Cells are incubated for 1-2 h before they are scored. Cells that take up the vital stain are scored as 'live'.
Data collected by investigator
• Susceptibility to Bacillus anthracis lethal factor (S=susceptible, R=resistant)
anthrax: is an infectious disease caused by bacteria called Bacillus anthracis. Infection in humans most often involves the skin, the gastrointestinal tract, or the lungs. Pathogenesis depends on secretion of three factors that combine to form two bipartite toxins (edema toxin and lethal toxin).
anthrax toxins: encoded polypeptides that are required for the virulence of B. anthracis, namely called protective antigen (PA), edema factor (EF) and lethal factor (LF).
edema toxin: consists of protective antigen (PA) and edema factor (EF), which causes edema associated with cutaneous infections.
lethal toxin: consists of protective antigen (PA) and lethal factor (LF), which causes death in systemic infections.
resistant macrophage cells: show more than 90% viability when treated with 10,000 ng/mL lethal toxin.
susceptible macrophage cells: show almost complete lethality (less than 10% viability) when treated with 10,000 ng/mL lethal toxin.
References
Dietrich WF. Using mouse genetics to understand infectious disease pathogenesis. Genome Res. 2001 Mar;11(3):325-31.
PubMed 11230157