| Xie Q, Matsunaga S, Shi X, Ogawa S, Niimi S, Wen Z, Tokuyasu K, Machida S.
(2003)
Protein Expression and Purification,
32,
68-74 |
| Structure-Function |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 4h |
| pET28a |
| An overnight culture of LB broth grown at 37degC was diluted 50-fold with fresh LB broth and incubated at 37degC. When OD600 reached 0.6, expression was induced with 1M IPTG and cells were grown a further 4h.
|
| Not Stated |
| OD 600 =
0.6 |
| Sonication |
| None |
| None |
| not stated |
| Dilution |
| n/a |
| 6M GdnHCl, PBS pH 7.4, 40mM DTT |
| CTAB: 0.1% cetyltrimethylammoniun bromide buffer containing 2mM DL-cystine/PBS |
| None |
| no |
| 7.4 |
| 25.0 |
|
| 2h |
| DL-cystine |
| n/a,2mM |
| Inclusion bodies were suspended in PBS(-) and the volume was adjusted so that the protein concentration was 28 mg/ml. Then, the inclusion bodies were completely unfolded in solubilization buffer for 1 h at 25degC. Usually 150microlitres of inclusion body suspension was used for unfolding. A mixture of 450microL of 8 M GdnCl with 6microL of 4 M DTT was added to 150microL of inclusion body suspension. A 70-fold volume of freshly prepared cetyltrimethylammonium bromide (CTAB) buffer (containing 0.1% CTAB, 2 mM -cystine/PBS(-), pH 7.4) was added to the unfolded protein solution and the mixture (42 ml) was allowed to stand at 25degC for 1 h. Next, 10.5 ml of 3% CA solution (final concentration, 0.6%) was added to refold the domain. After further incubation for 1 h at 25degC, the solution was centrifuged at 20,000g for 10 min and the resulting supernatant was used as the refolded domain.
Ten milliliters of 50% nickel-nitrilotriacetic acid (Ni-NTA) resin slurry was added to the refolded protein solution and mixed gently by rotation for 30 min to allow the refolded domain to bind to the resin. This resin with immobilized refolded protein was then packed into a column. After the column was washed with PBS(-) containing 0.1 M imidazole, the refolded domain was eluted by a gradient of imidazole (0.1-0.5 M) using the FPLC system (Amersham-Pharmacia Biotech, Sweden). Fractions showing absorbance at OD280 were collected and an aliquot of each was applied to SDS-PAGE. Fractions containing protein at the expected molecular weight of CTLD were pooled and dialyzed against PBS(-). The protein concentration at each step was determined by a sodium deoxycholate-trichloroacetic acid protein precipitation techniques. |
| Far-UV Circular Dichroism |
| None |
| None |
|
| 27mg/L |
|
| C-lectin like domain (aa144-273) cloned and refolded
No disulfides
mgsshhhhhhssglvprgshmasmtggqqmgr
CPQDWIW 150
HGENCYLFSS GSFNWEKSQE KCLSLDAKLL KINSTADLDF IQQAISYSSF 200
PFWMGLSRRN PSYPWLWEDG SPLMPHLFRV RGAVSQTYPS GTCAYIQRGA 250
VYAENCILAA FSICQKKANL RAQ
Number of amino acids: 162
Molecular weight: 18254.6
Theoretical pI: 8.80
|