| Lalitha PV, Ware LA, Barbosa A, Dutta S, Moch JK, Haynes JD, Fileta BB, White CE, Lanar DE.
(2004)
Infection and Immunity,
72,
4464-70 |
| Vaccine studies |
| N-terminal +C terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 2.5 h |
| pCR2.1 |
| For expression in shake flasks, cells were grown to an optical density at 600 nm of 0.5 and then induced with a final concentration of 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Cells were harvested after 2 h of induction by centrifugation at 6,200 × g and 4°C for 30 min. Expression in a 10-liter bioreactor (New Brunswick Scientific, Edison, NJ) used Terrific Broth media (1.2% tryptone, 2.4% yeast extract, 72 mM K2HPO4, 28 mM KH2PO4 [pH 7.2]) containing 0.8% glycerol and 100 μg of ampicillin per ml. The 10 liters of media in the bioreactor was inoculated with 100 ml of an overnight culture, and growth was allowed to proceed at 37°C. pH 7.2 was maintained by controlled addition of either HCl or NaOH. At an optical density at 600 nm of 7 to 8, the temperature was reduced to 25°C and then IPTG was added to a final concentration of 0.5 mM. At 2 h after induction, cells were harvested by centrifugation at 5,000 × g and 4°C for 30 min. Under these conditions, all six constructs had similar growth characteristics. The cell pastes were stored at −80°C. Aliquots were taken from these for developing the purification conditions for each of the subdomain proteins. |
| IPTG |
| OD 7-8 =
600 |
| high-pressure microfluidizer |
| None |
| Ni-NTA agrose chromatography |
| insoluble |
| Dilution |
| n/a |
| 15 mM Na2HPO4, 5.1 mM KH2PO4, 450 mM NaCl, and 2.5% sodium N-lauroylsarcosine (Sarkosyl) (pH 7.4) |
| 30 to 40 μg/ml in a redox-coupled GSH-GSSG solution |
| Ni-NTA agrose chromatography |
| no |
| 8.0 |
| 25.0 |
| n/a |
| overnight |
| GSH/GSSG |
| n/a |
| Purification of the subdomain fragments. The E. coli cell pastes were solubilized in a buffer containing 15 mM Na2HPO4, 5.1 mM KH2PO4, 450 mM NaCl, and 2.5% sodium N-lauroylsarcosine (Sarkosyl) (pH 7.4) at a concentration of 10 ml of buffer/g of cell paste. The cells were disrupted by a single pass through a high-pressure microfluidizer (model 1109; Microfluidic Corp., Newton, Mass.). Following clarification by centrifugation (12,000 × g, for 45 min at 4°C), supernatants containing the recombinant proteins were incubated by a batch method with nickel nitrilotriacetic acid (Ni-NTA)-agarose chelating resin (0.4 ml of resin/g of cell paste) (Qiagen Inc.) at room temperature (RT) (~22°C) for 1 h in the presence of 35 mM imidazole. The resin was then loaded into a fritted column, and the unbound proteins were allowed to flow through. The resin was washed with a minimum of 40 column volumes (cv) of 20 mM NaHPO4-450 mM NaCl-10 mM imidazole-0.125% Sarkosyl (pH 7.4) followed by 10 cv of 20 mM NaHPO4-15 mM imidazole (pH 8.0). Bound proteins were eluted with 500 mM imidazole in 20 mM phosphate buffer containing 0.125% Sarkosyl (pH 8.0). Proteins eluted from the Ni-NTA-agarose were rapidly diluted to 30 to 40 μg/ml in a redox-coupled GSH-GSSG solution and allowed to fold overnight as described previously (7). After the refolding period, the protein solution was pH adjusted (to 5.8 for D I and D II, 6.4 for D III, and 6.0 for all double domains) and passed through preequilibrated SP Sepharose (Amersham Pharmacia Biotech) (~0.3 ml of resin/g of cell paste). The resin-bound protein was washed with a minimum of 50 cv of 20 mM NaHPO4-1mM EDTA containing 100 mM NaCl (for double-domain constructs) or 250 mM NaCl (for single-domain constructs) at pH 6.0. The bound protein was eluted with 20 mM NaHPO4-1 mM EDTA (pH 7.4) plus 150 mM NaCl (pH 6.0) (for single-domain constructs) 300 mM NaCl (pH 6.0) (for D I+II), or 400 mM NaCl (pH 6.0) (for D I+III and D II+III). |
| SDS-PAGE |
| None |
| None |
| n/a |
| 15 to 20 mg/L |
| n/a |
| n/a |