| Dutta S, Lalitha PV, Ware LA, Barbosa A, Moch JK, Vassell MA, Fileta BB, Kitov S, Kolodny N, Heppner DG, Haynes JD, Lanar DE.
(2002)
Infection and Immunity,
70,
3101-3110 |
| Identification and Characterization |
| N-terminal +C terminal hexahistidine tag + thioredoxin tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| origami B(DE3) |
| 27.0 |
| 1 h |
| pET32 |
| he expression of r-AMA1/E protein was carried out in a 10-liter bioreactor (New Brunswick Scientific, Edison, N.J.) at the lab scale and in a 300-liter bioreactor (New Brunswick Scientific) at the Walter Reed Army Institute of Research Pilot Bioproduction Facility. Medium consisting of Super Broth containing 0.8% glycerol and 12.5 µg of tetracycline ml-1 was inoculated with a 3-liter overnight culture started from production seed lot no. 0788. The bioreactor temperature was maintained at 27°C (pH 7.2), and agitation was maintained at 400 rpm. At a cell density for which the optical density at 600 nm (OD600) was 7.0, IPTG was added to a final concentration of 0.1 mM. One hour later, cells were harvested by centrifugation and frozen at -80°C. Aliquots (10 to 150 g) from this production were used to develop a purification and refolding process of r-AMA1/E protein at the lab prior to scale-up (1,500 g) in a GMP environment. |
| IPTG |
| OD 7.0 =
600 |
| Microfluidizer |
| Detergents |
| Metal affinity chromatography |
| insoluble |
| Dilution |
| buffer A(15 mM Na2HPO4, 5.1 mM KH2PO4, 450 mM NaCl) with 10 mM imidazole and 0.125% Sarkosyl; pH 7.4 |
| 20 mM sodium phosphate, 25 mM imidazole, 0.125% Sarkosyl; pH 8.0 |
| 20 mM sodium phosphate, 1 mM EDTA, 1 mM reduced glutathione [GSH], 0.25 mM oxidized glutathione [GSSG]; pH 8.0 |
| Metal affinity chromatography |
| no |
| 8.0 |
| 22.0 |
| n/a |
| 15 h |
| GSH/GSSG |
| 1/0.25 mM |
| Refolding. The Ni2+ elution was diluted 40-fold (vol/vol) rapidly in degassed buffer E (20 mM sodium phosphate, 1 mM EDTA, 1 mM reduced glutathione [GSH], 0.25 mM oxidized glutathione [GSSG]; pH 8.0). The refolding buffer was prepared fresh, and refolding was carried out at room temperature (22°C) for a minimum of 15 h under nitrogen. The final protein product resulting from this refolding protocol was referred to as AMA1/E. Several other variations to the refolding protocol described above were also tested. One such variation included reduction of the Ni2+-eluted proteins with 5 mM dithiothreitol (DTT) for 1 h at 37°C before refolding. The protein that was yielded after reduction and refolding followed by ion-exchange purification was referred to as RR-AMA1/E.
Ion-exchange purification. Ion-exchange column resins were sanitized with 0.2 N NaOH before use and then equilibrated to the initial binding conditions. After the refolding step, AMA1/E protein was concentrated on a DEAE Sepharose anion-exchange column (Amersham Pharmacia Biotech) (0.25 ml of packed resin per g of paste) and the column was pre-equilibrated with buffer E without the GSH or GSSG. After the protein was loaded, the column was washed with a minimum of 30 CV of the same equilibration buffer followed by 10 CV of buffer F (5 mM sodium phosphate, 50 mM NaCl, 1 mM EDTA; pH 8.0). AMA1/E was eluted in buffer F containing a final concentration of 100 mM NaCl (pH 8.0). AMA1/E eluted from the DEAE column was pH adjusted to 5.7 by the addition of 1 M NaH2PO4 · H20 and loaded on an SP Sepharose cation-exchange column (Amersham Pharmacia Biotech) (0.15 ml of packed resin per g of paste) pre-equilibrated with buffer G (50 mM sodium phosphate, 0.1 mM EDTA, 100 mM NaCl; pH 5.7). The column was washed with 20 CV of buffer G containing a final NaCl concentration of 275 mM (pH 5.7) followed by 10 CV of a pH exchange buffer (5 mM sodium phosphate, 0.1 mM EDTA; pH 7.1). AMA1/E was eluted from the column in formulation buffer (23.5 mM NaH2PO4 · H2O, 37.5 mM NaCl, 0.1 mM EDTA; pH 7.1). |
| postrefolding'analysis |
| None |
| None |
| n/a |
| 0.75 to 1 mg/l |
| >99% |
| n/a |