| He HW, Feng S, Pang M, Zhou HM, Yan YB.
(2007)
Int J Biochem Cell Biol,
39,
1816-1827 |
| Folding |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3)pLysS |
| 37.0 |
| 14 h |
| pET21b |
| Protein expression and purification
The gene of wild-type (WT) rabbit MM-CK and the pET-21b expression vector used for site-directed mutagenesis are described elsewhere (Zhao et al., 2006). WT-CK and the mutants were expressed in Escherichia coli BL21[DE3]-pLysS (Stratagene, Heidelberg, Germany) and purified as described previously (Feng, Zhao, Zhou, & Yan, 2007; Zhao et al., 2006).
Expression of MM-CK
The recombinant plasmid pET21-CK was transformed into E. coli BL21(DE3) for expression of the CK protein. Positive colonies were identified by PCR. A fresh, isolated colony was chosen and incubated overnight in liquid LB medium at 37 °C. The overnight culture was diluted 1:10 in the same LB medium and grown at 37 °C until A600 reached 0.6–0.8 absorbance units. The expression was then induced at 25 °C for about 14 h by the addition of 0.4 mM of isopropyl-β-D-thiogalactopyranoside (IPTG).
|
| IPTG |
| OD 0.6-0.8 =
600 |
| ultrasonication |
| None |
| Ion-exchange chromatography |
| insoluble |
| Dilution |
| n/a |
| GdnHCl concentrations ranging from 0 to 3 M in 10 mM Tris–HCl buffer |
| 10 mM Tris–HCl buffer, pH 8.0, containing 0.1 M GdnHCl |
| Ion-exchange chromatography |
| no |
| 8.0 |
| 25.0 |
| n/a |
| n/a |
| None |
| n/a |
| Purification of MM-CK
The recombinant cells were collected by centrifugation (5000 × g, 10 min) and resuspended in lysis buffer (10 mM Tris–Cl, pH 8.0; 0.1 mM dithiothreitol (DTT); 0.1 mM phenylmethanesulfonyl fluoride (PMSF)). The cells were lysed by ultrasonication and the debris was removed by centrifugation (15 000 × g, 20 min, 4 °C). The supernatant was loaded on a Blue Sepharose CL-6B column preequilibrated with buffer containing 10 mM Tris–acetate, pH 8.0 and 0.1 mM DTT. The active fractions flowing through the above column were pooled and then loaded on a preequilibrated DEAE-Fast Flow column for ion-exchange chromotography. The column was washed with four to five column volumes of 10 mM Tris–Cl, pH 8.0 and 0.1 mM DTT before the CK was eluted with gradient elution using 0–0.5 M NaCl. The purified CK presented a single band on SDS-PAGE. All purification steps were carried out at 4 °C. Protein concentration was determined by the Bradford method [24] with bovine serum albumin as the standard.he refolding was initiated by a 100-fold dilution of the denatured CK into 10 mM Tris–HCl buffer, pH 8.0, containing 0.1 M GdnHCl. |
| Activity assay |
| None |
| None |
| n/a |
| n/a |
| n/a |
| The purification protocol is from S.Y. Guo, Z. Wang, S.W. Ni and X.C. Wang, Consequences of a six residual deletion from the N-terminal of rabbit muscle creatine kinase, Biochimie 85 (2003), pp. 999-1005 |