| Koo HM, Kim JH, Hwang IK, Lee SJ, Kim TH, Rhee KH, Lee ST.
(2002)
Mol Cell,
13,
118-124 |
| Protein refolding |
| C-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 6 h |
| pET22b |
| The resultant expression construct pET22b-chMT1-MMP-(His)6 was transformed into an E. coli strain BL21(DE3). The transformed cells were grown in a Luria broth that contained 100 ug/ml ampicillin at 37c to a cell density of A600==0.6. The expression of recombinant protein induced in the presence of 1 mM IPTG at 37c for 6 h. The cell pellet was resuspended in a 1/10 culture volume of 50 mM Tris-HCl, pH 8.0, 2 mM EDTA. The resuspended cells (to which lysozyme and Triton X-100 were added to concentrations of 100 ug/ml and 0.1% respectively) were incubated at 30c for 15 min. The cell lysate was sonicated until it lost its viscosity, then centrifuged at 12,000 for 15 min at 4c in order to separate supernatants (soluble fraction) and pellets ( insoluble fraction). |
| IPTG |
| OD 0.6 =
600 |
| Sonication |
| Lysozyme |
| Ni-NTA column |
| insoluble |
| Dialysis |
| 50 mM Tris-HCl, pH 8.0, 2 mM EDTA |
| 50 mM Tris-Hcl, pH 8.5, 6M urea, 30 mM 2-mercuptoethanol |
| 50 mM Tris-Hcl, pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.5 mM ZnCl2 |
| Ni-NTA column |
| yes |
| 7.5 |
| 0.0 |
| n/a |
| n/a |
| None |
| n/a |
| The insoluble fraction was solubilized in a resuspending buffer (50 mM Tris-Hcl, pH 8.5, 6M urea, 30 mM 2-mercuptoethanol). The solubilized protein were loaded onto a Ni2-NTA affinity column that was equilibrated with resuspended buffer . The chMT1-MMP polypeptide, the eluent was diluted to 100 ug/ml and 2-mercaptoehtanol was added up to 150 mM. The protein was gradually dialyzed with the refolding buffer(50 mM Tris-Hcl, pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.5 mM ZnCl2). |
| Western Blot |
| None |
| None |
| n/a |
| 30% |
| n/a |
| The refolding temperature not stated |