| matrix assisted refolding/ renaturing gel filtration |
| 50 mM NaP pH 8.0, 7.0 M GdmCl, 10 mM imidazole, 2.5 mM TCEP |
| 100 mM NaP pH 8.0, 7.0 M GdmCl, 10 mM imidazole |
| 50 mM NaP pH 7.8, 100 mM NaCl, protease inhibitor cocktail (Complete EDTA-free, Roche) |
| IMAC |
| no |
| 7.8 |
| 25.0 |
|
|
| TCEP (facultative) |
| n/a |
| All SlyD*/FkpA fusion proteins were purified by using virtually the same protocol. E. coli BL21(DE3) cells harboring the particular pET24a expression plasmid were grown at 37°C in LB medium plus kanamycin (30 µg/ml) to an OD600 of one, and cytosolic overexpression was induced by adding isopropyl-ß-D-thiogalactoside to a final concentration of 1 mM. 3 h after induction, cells were harvested by centrifugation (20 min at 5000 x g), frozen and stored at –20°C. For cell lysis, the frozen pellet was resuspended in chilled 100 mM sodium phosphate pH 8.0, 7.0 M GdmCl, 10 mM imidazole and the suspension was stirred for two hours on ice to complete cell lysis. After centrifugation and filtration (cellulose nitrate membrane, 0.45 µm/0.2 µm), the lysate was loaded on a Ni-NTA column equilibrated with the lysis buffer. The subsequent washing step was varied from 10 mM imidazole for the FkpA-X- type fusion proteins to 25 mM imidazole for the SlyD*-X type fusion proteins. At least 10-15 column volumes of washing buffer were applied. Then, the GdmCl solution was replaced by 50 mM sodium phosphate pH 7.8, 100 mM NaCl to induce refolding of the matrix bound protein. In order to avoid reactivation of copurifying proteases, a protease inhibitor cocktail (Complete®, EDTA-free, Roche) was included in the refolding buffer. 15 - 20 column volumes of refolding buffer were applied in an overnight reaction. The native fusion protein was then eluted by 250 mM imidazole in 50 mM sodium phosphate pH 7.8, 100 mM NaCl. Protein containing fractions were assessed for purity by SDS-PAGE and pooled. Finally, the proteins were subjected to size exclusion chromatography (Superdex 200 HiLoad, Amersham Pharmacia) and the protein containing fractions were pooled and concentrated in an Amicon cell (YM 10).
Alternatively, the fusion proteins were eluted from the Ni-NTA column by 7.0 M GdmCl in 50 mM sodium phosphate pH 2.5 after the washing step. Protein containing fractions were pooled and subjected to renaturing gel filtration on a Superdex 200 HiLoad column preequilibrated with 50 mM sodium phosphate pH 7.5, 100 mM NaCl, 1 mM EDTA. 5 ml of the protein (5-20 mg/ml) in 7.0 M GdmCl were applied on the column and the elution of the renatured protein was monitored at 280 nm.
|
| UV spectrum/Near UV CD/ Immunology |
| None |
| None |
| NULL |
| > 20 mg gp36 fusion protein/g E. coli wet weight |
| > 95 % (Coomassie-stained SDS-PAGE) |
| The soluble gp36 ectodomain variants comprise the aggregation-prone gp36 fragment 534-676 and one or two units of FkpA or SlyD, respectively, as solubility-confering fusion partners. The chaperones are fused to the N-terminus of the target molecule via an extended flexible linker rich in glycine and serine residues. |