Refolding Record:
| Protein | |
|---|---|
| Protein Name | CHA-II protein |
| Abbreviated Name | CHA-II |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Cepaea hortensis |
| UniProt Accession | Q5F4K1 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 101 |
| Molecular Weight | 11238.5 |
| Pi | 8.09 |
| Molecular Weight | 11238.5 |
| Disulphides | Unknown |
| Full Sequence |
AQTGEIDYGSDSSWPKVPSDDPNKDRTRELVKNVTFPSPYCRPPKVILSVTTLDSDHTKNLRYQARLKSVSPSGFSASSYTWHDTTIYRMKLSWLAVED
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Gerlach D, Schlott B, Zähringer U, Schmidt KH. (2005) FEMS Immunol Med Microbiol., 43, 223-232 |
| Project Aim | Expression and charactrization |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15 |
| Expression Temp | 37.0 |
| Expression Time | 20 h |
| Expression Vector | pQE-30 |
| Expression Protocol | A 2 ml LB over night culture of the E. coli clone pQE30-rCHA-II was used for inoculation of 200 ml LB medium containing 100 lg/ml ampicilline and 25 lg/ml kanamycine. After shaking at 37 °C for 3 h the lectin production was induced by addition of IPTG to a final concentration of 2 mM. Incubation was continued for 20 h. The cells were harvested by centrifugation, washed once with PBS and suspended in 15 ml buffer I (0.15 M NaCl, 0.02 M Tris–HCl, pH 7.7). The suspension was sonicated three times for 7 min at 4 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | PBS |
| Solubilization Buffer | 6 M guanidine hydrochloride buffer (GuHCl, pH 7.5) |
| Refolding Buffer | 10 mM GlcNAc in 0.02 M Tris–HCl buffer (pH 8) |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The inclusion bodies were dissolved in 10 ml 6 M guanidine hydrochloride buffer (GuHCl, pH 7.5) and used for affinity chromatography on a Ni–agarose column. After washing with a buffer containing 6 M GuHCl plus 20 mM imidazol–HCl (pH 7.0), the lectin was eluted with 0.5 M imidazol (pH 7.0) in 6 M GuHCl. For analysis of eluted fractions, small samples were precipitated with 4 volumes ethanol and the precipitates were dissolved in sample buffer and analysed in SDS–PAGE. Lectin containing fractions were combined. The lectin was precipitated by dialysis against 0.02 M Tris–HCl buffer (pH 7.7) and dissolved in 6 M urea and 0.02 M Tris–HCl buffer (pH 7.7) containing 5 mM dithiothreitol (DTT). After dilution of 1:5 in PBS, this solution of recombinant lectin (rCHA-II) was directly used for haemagglutination. Refolding of rCHA-II dissolved in 6 M urea (about 1 mg/ml) has been done by a fast dilution into 15 volumes 10 mM GlcNAc in 0.02 M Tris–HCl buffer (pH 8) and incubation over night at 4 °C. The refolded lectin (yield about 10%) was concentrated by precipitation at 80% ammonium sulfate saturation and dissolved in water. The refolded protein was stable and remained soluble in water or PBS at 4 °C and could be used in immunodiffusion on agar gels. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 10% |
| Purity | n/a |
| Notes | n/a |