| Hatakeyama T, Shiba K, Matsuo N, Fujimoto T, Oda T, Sugawara H, Aoyagi H.
(2004)
J Biochem.,
135,
101-107 |
| Purification & characterization |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3)pLysS |
| 0.0 |
| 00 |
| pET-3a |
| The nucleotide sequence of the rCEL-I gene was confirmed by sequencing with a Hitachi DNA sequencer SQ5500E. The inserted rCEL-I gene was digested with NdeI and BamHI, and then ligated with the pET-3a vector (Novagen) previously digested with the same enzymes. Since the recombinant protein was expressed in E. coli BL21(DE3)pLysS (Novagen) as inclusion bodies, they were collected by centrifugation from the cells after sonication in TBS, and immediately solubilized with 20 mM Tris-HCl buffer, pH 8.5, containing 8 M urea and 1% 2-mercaptoethanol. |
| IPTG |
| OD n/a =
n/a |
| Sonication |
| None |
| DEAE-Cellulofine column chromatography |
| insoluble |
| Dialysis |
| n/a |
| 20 mM Tris-HCl buffer, pH 8.5, containing 8 M urea and 1% 2-mercaptoethanol |
| Tris-buffered saline |
| DEAE-Cellulofine column chromatography |
| no tag |
| 0.0 |
| 0.0 |
| n/a |
| 3 day |
| None |
| n/a,n/a |
| The solubilized protein was directly applied to a DEAE-Cellulofine column (3 x 3 cm) in 20 mM Tris-HCl, pH 8.5, containing 4 M urea, and eluted with a NaCl gradient, 0 to 0.5 M. The rCEL-I fractions were pooled and dialyzed against TBS for three days. The active protein exhibiting carbohydrate-binding activity was purified by affinity chromatography on a GalNAc-Cellulofine column (1.5 x 3 cm) (6). The rCEL-I dimer was finally purified by gel filtration on a Sephadex G-50 column (2.5 x 46 cm) in TBS.Purification of Native CEL-I— Native CEL-I was purified from the protein extract of C. echinata by column chromatography on lactosyl-Cellulofine, GalNAc-Cellulofine, and Sephadex G-75, essentially as reported earlier (6). |
| Haemagglutination assays |
| None |
| None |
| n/a |
| 20 mg/l |
| n/a |
| Refolding temperature and pH are not stated |