| Boyington, J. C., Raiz, A. N., Brooks, A. G., Patamawenu, A., Sun, P. D.
(2000)
Protein Expression and Purification,
18,
235-241 |
| Structural Studies,Recombinant Protein Expression |
| N-terminal hexahis + pro seq |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 5h |
| pET30a |
| 150ml overnight culture was used to inoculate 10 L fermentor vessel that contained 9L of Super-Broth, 450mg o kanamycin, and 1 mL antifoam 289. Cells were induced and harvested after incubation by centrifugation. Harvested cells were resuspended in lysis buffer (25%sucrose, 50mM TrisHCl, pH 8.0) and were subjected to cell disruption. DNase-1 was added to remove residual DNA. |
| IPTG |
| OD 595 =
1.5 |
| Freeze/Thaw+Sonication |
| Chemicals |
| Washing inclusion body |
| insoluble |
| Dilution/Dialysis combination |
| 2M urea, 5mM EDTA, 5mM DTT, 0.1% Triton X-100, and 0.1M TrisHCl, pH 7.9 |
| 6M GuHCl |
| 1M Arginine, 0.5 M NaCl, 25 mM CaCl2, 5mM cystamine, 1mM NiCL, 50mM TrisHCl, pH 8.0 |
| Washing inclusion body |
| no |
| 8.0 |
| 4.0 |
| n/a |
| 24-48h |
| None |
| n/a |
| Inclusion bodies were washed several times with wash buffer and then dissolved in 120 mL of solubilization buffer. The solubilized protein was then injected into 2L refolding buffer, using a 19 gauge needle in three equal aliquots at 6h intervals with vigorous stiring. The refolding mixture was then stirred moderately for 24-48h at 4degC and then dialyzed against water at 4deg until final salt content was less than 30 mM. Dialyzed refolding reaction mixture was then centrifuged(27,500g, 30min)to remove aggrigates and then applied to a Mono P column. Bound CD94 was eluted with a 60 ml elution gradient from 10 mM HEPES, pH 7.00. 400 mM NaCl, 10 mM Acetate, pH 4.4 Eluted samples were again dialyzed against 5 mM TrisHCl, pH 8.0 |
| SDS-PAGE |
| None |
| L-Arginine |
| 1M |
| n/a |
| n/a |
| n/a |