| Ye QZ, Johnson LL, Hupe DJ, Baragi V
(1992)
Biochemistry,
31,
11231-11235 |
| Crystallography,NMR,Structure-Function |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| DH5alphaF(IQ) |
| 37.0 |
| 6h |
| pGEMEX-1 |
| 2L of 2TY medium was inoculated with 20ml overnight culture. Cells were grwon at 37degC unatil OD600 reached 0.8. Expression was induced by the addition of the phage M13/T7 (plaque forming unit per mL=1 x 10e11, multiplicity of infection=10) and 1mM IPTG. Cells were then grown for a further 6h. |
| IPTG |
| OD 600 =
0.8 |
| French Press |
| None |
| Washing inclusion body |
| insoluble |
| Dilution |
| 50mM TrisHCl pH 7.6 |
| 8M GdnHCl |
| 50mM TrisHCl pH 7.6, 10mM CaCl2, 0.1mM ZnCl2, 1microg/ml each of leupeptin, aprotinin, pepstatin |
| Washing inclusion body |
| no tag |
| 7.6 |
| 4.0 |
| n/a |
| n/a |
| None |
| n/a |
| Cells (5.97g wet weight) were resuspended in 25ml of 50mM TrisHCl pH 7.6 and lysed by two passages through a French press at 14000psi. The pellet obtained after centrifugation (20000g, 30min) was washed twise with wash buffer and then solubilized wtih 10-ml of 8M GdnHCl. The mixture was centrifuged (20000g, 20min) and the supernatant was then added drop-wise to 100ml refolding buffer stirred at 4degC. The refolded solution was centrifuged (20000g, 20min), the pellets were dissolved in another 10ml 8M GdnHCl and refolding was repeated twice. The supernatants of the three refoldings were combined, ammonium sulfate was added to 20% saturation and the protein was then purified using phenyl-sepharose and Q-sepharose columns. |
| Enzyme activity,Amino acid sequencing |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |