| Brown, L. R., Deng, J., Noll, D. M., Mori, N., Clarke, N. D.
(1997)
Protein Expression and Purification,
9,
337-345 |
| Recombinant Protein Expression |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 4h |
| pG5 |
| Cells were resuspended in 50ml of 20M TrisHCL, pH 7.5 containing 1 mM EDTA and 20% sucrose incubated on ice for 10min. This solution was centrifuged(4000g) and the pellet resuspended in 50ml ice water prior to another round of centrifugation(8500g, 20min) to yield spheroplasts. Spheroplasts were resuspended in 10ml PBS with 5mM EDTA, 5mM Mg, 0.5 PMSF and lysed. The homogenate was incubated for 10 min at room temp with 1300U ribonuclease T1 and 4000microgram deoxyribonuclese I. Solution was then diluted with 40ml PBS with 5mM EDTA, 5mM Mg2+ and centrifuged(13,000g, 30min). Inclusion bodies were resuspended in 50ml washing buffer and homogenized to resuspend completely, this was incubated on ice for 10min and then centrifuged(25,000, 20min) |
| IPTG |
| OD 600 =
0.6-0.8 |
| High pressure homogenization |
| Lysozyme |
| Washing inclusion body |
| insoluble |
| Dilution |
| PBS with 5mM EDTA, 25% sucrose, 1% Triton X-100 and 0.5mg/ml lysozyme |
| 50mM Tris-HCL, pH 9.0, 5M Guanidine hydrochloride, 5mM EDTA, 1mM DDTT |
| 50mM glycine, pH 9.0, 1 mM EDTA, 1mM DTT and 20% glycerol |
| Washing inclusion body |
| no tag |
| 9.0 |
| 4.0 |
| n/a |
| overnig |
| DTT |
| 1mM |
| Inclusion bodies were denatured with 10ml of solubilization buffer and incubated on 1h and centrifuged(12,500g, 30min). Protein was renatured by 100-fold dilution with refolding buffer with gentle stiring overnight. |
| Enzyme activity |
| None |
| Glycerol,Glycine |
| n/a |
| n/a |
| n/a |
| n/a |