| Grimm KM, Trigona W, Heidecker GJ, Joyce JG, Fu T-M, Shiver JW, Keller PM, Cook JC
(2001)
Protein Expression and Purification,
2001,
270-281 |
| Functional Studies |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| Xa90 |
| 37.0 |
| 3h |
| pHN1+ |
| Cells were induced with 0.4mM IPTG during early log phase, then grown for 3h, harvested by centrifugation (7100g, 10min) and stored at -70degC. |
| IPTG |
| OD =
|
| Sonication |
| Lysozyme |
| None |
| insoluble |
| Dilution with complex partner subunits |
| 0.5% Triton X-100, 50mM TrisHCl, 100mM NaCl, 1mM EDTA, 1mM DTT pH 8.0 |
| 25mM sodium Mes pH 6.0, 8M urea, 10mM EDTA, 0.1mM DTT |
| 400mM arginine, 100mM TrisHCl pH 8.3, 2mM EDTA |
| None |
| yes |
| 8.3 |
| 10.0 |
| 3microM |
| 35-50h |
| None |
| n/a |
| Frozen cells were thawed and resuspended in 50mM TrisHCl pH 8.0, 25% sucrose, 1.0% Triton X-100, 1mM EDTA, 0.1% sodium azide, 11mM DTT, 1mg/ml lysozyme, 5mM MgCl at a concentration of 1.3g wet cell weight/60ml. A 2mg/ml DNase I stock solution in 75mM NaCl, 50% glycerol was added to the cell suspension to a final concentration of 1.7%(v/v). The cell suspension was placed in an ice bath with stirring and was sonicated for 1.5min. The lysate centrifuged (11000g, 10min, 4degC). The pellet was resuspended in 40ml wash buffer, sonicated and centrifuged - this process was repeated a further three times, the third time using wash buffer without Triton X-100.Triton X-100 and centrifuged one last time. The pellet was then resuspended in 0.4mL sterile H2O and then dissolved in 5.0ml solubilization buffer.
Refolding was performed by diluting the protein with beta-2-microglobulin(b2m) and an antigen peptide into refolding buffer prechilled to 10degC. The concentration of the protein (Mamu-A 01) was ranged from 1-3microM and urea was kept below 0.3M. The molar ratio of Mamu-A 01) to b2m to peptide was 3:2:28. For 236ml of refolding buffer, 7.1mg of peptide was dissolved in 0.5ml H2O and added to the refolding buffer with vigorous stirring. 0.236 micromol of Mamu-A 01 and 0.4 micromol of b2m were each dissolved in 5.0ml of 3M GdnHCl, 10mM sodium acetate, 10mM EDTA pH 4.2 and added to the stirring folding reaction through 26-gauge needles. The folding reaction was covered and incubated without stirring for 14-20h at 10degC, and an additional 0.236mol Mamu-A 01 was added to the folding reaction. The folding reaction was incubated at 10degC for 7-10h, then a further 0.236 mol of Mamu-A 01 was added. The folding reaction was incubated at 10degC for a further 14-20h. Final concentrations were 3.0microM Mamu-A 01, 2.0microM b2m, 27.7microM peptide.
The folding reaction product was then concentrated and purified by size exclusion chromatography |
| SDS-PAGE |
| None |
| None,L-Arginine |
| 0.4M |
|
|
| More details of an "enhanced" process also detailed in the same publication - refolding method is essentially the same, but final concentrations in the refolding buffer are:
1.2microM Mam-A 01, 0.8microM b2m, 10.8microM peptide |