Refolding Record:
| Protein | |
|---|---|
| Protein Name | Osmotin-like protein |
| Abbreviated Name | SnOLP |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Solanum nigrum (Black nightshade) |
| UniProt Accession | Q8SA57 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 247 |
| Molecular Weight | 26814.0 |
| Pi | 6.52 |
| Molecular Weight | 26814.0 |
| Disulphides | Unknown |
| Full Sequence |
MGYLRSSFVFFLLTFVTYTYATSFEVRNNCPYTVWAASTPIGGGRRLDRGQTWVINAPRGTSMARIWGRTNCNFDGAGRGSCQTGDCGGVLQCTGWGKPPNTLAEYALNQFSNLDFWDISLVDGFNIPMTFAPTNPSGGKCHSIQCTANINGECPAALRVPGGCNNPCTTFGGQQYCCTQGPCGPTELSKFFKQRCPDAYSYPQDDPTSTFTCPSDSTNYRVVFCPNGVTSPNFPLEMPSSTDEVAK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Campos MD, Silva MS, Magalhaes CP, Ribeiro SG, D Sarto RP, Vieira EA, Grossi de Sa MF. (2008) Microb Cell Fact., 7, 1 |
| Project Aim | Protein refolding |
| 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 | 3 h |
| Expression Vector | pQE30-SnOLP |
| Expression Protocol | Overexpression of His6-tagged mature SnOLP in E. coli Preliminary experiments with the pQE30-SnOLP expression construct were performed to determine the solubility of the SnOLP, according to the instructions of manufactures (QiaEXpressionist-QiaGen), and to establish the expression levels of the mature protein. Cells of E. coli M15 strain carrying out the construct were cultured overnight at 37 °C in 5 mL of Luria-Bertani (LB) medium containing 100 µg/mL ampicillin and 25 µg/mL kanamycin (i.e. LB selective medium) under vigorous agitation (200 rpm). This pre-inoculum suspension was then used to inoculate 500 mL fresh LB selection medium, which was agitated until an O.D. 600 of 0.6 was reached. Thereafter, an aliquot of non induced control cells was colleted and reserved, and the expression of His6-tagged mature SnOLP protein was induced in the left cells by addition of IPTG to a final concentration of 0.4 mM. The cells were cultivated at 37 °C for 3 h in the induction medium (i.e. LB selective medium plus IPTG) and afterwards collected by centrifugation at 4.000 g at 4 °C for 20 min. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Detergents |
| Pre-Refolding Purification | Metal affinity chromatography/ion exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 0.1 M Tris.HCl, 6 M Urea, 20 mM Imidazol, pH 8.0 |
| Solubilization Buffer | 0.1 M Tris.HCl, 6 M Urea, pH 8.0 |
| Refolding Buffer | 20 mM Tris.HCl pH 7.5, 500 mM NaCl, 10 mM reduced glutathione, 1 mM oxidized glutathione, and 20 % (w/v) glycerol |
| Pre-Refolding Purification | Metal affinity chromatography/ion exchange chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 10/1 mM |
| Refolding Protocol | Purification of the His6-tagged mature SnOLP protein As an attempt to determine the solubility of the protein, an aliquot of soluble fraction was recovered after centrifugation of cellular lysate, obtained from an aliquot of induced pelleted cells by using lysis buffer (50 mM potassium phosphate buffer pH 7.8, 400 mM NaCl, 100 mM KCl, 0.5% Triton X-100, 10% glycerol, 10 mM Imidazole), under native conditions. For a larger scale experiment, the pelleted cells were resuspended in denaturing lysis buffer (0.1 M Tris.HCl, 6 M Urea, pH 8.0) and the cellular suspension was maintained under gentle shaking for 1 hour at room temperature. Cellular debris were removed by centrifugation at 10.000 g for 25 min at 4 °C in order to obtain the solubilized fraction from the supernatant. Final purification of His6-tagged mature SnOLP was performed by high-performance immobilized-metal ion affinity chromatography (IMAC) on 10 mL batches of 50% Nickel-nitrilotriacetic-acid (Ni- NTA) resins (QiaEXpressionist-QiaGen), under constant shaking for 1 hour. In order to eliminate proteins nonspecifically bound to the column, the resin was submitted to the washing solution (0.1 M Tris.HCl, 6 M Urea, 20 mM Imidazol, pH 8.0) and afterwards the recombinant protein was eluted in two steps by using 10 mL of 0.1 M Tris.HCl, 6 M Urea, 200 mM Imidazol, pH 8.0. The eluted fractions were collected and reserved until use. Refolding of recombinant SnOLP Renaturation of solubilized mature His6-SnOLP was performed by dropwise mixing purified SnOLP protein under denaturing conditions into refolding buffer (20 mM Tris.HCl pH 7.5, 500 mM NaCl, 10 mM reduced glutathione, 1 mM oxidized glutathione, and 20 % (w/v) glycerol), at 4 °C, under stirring, in a protein:buffer ratio of 1:10. After repeated dialysis processes, at 4 °C, performed with deionized water to slowly remove denaturants, the recombinant protein was concentrated by lyophilization. Lyophilized recombinant SnOLP was resuspended in water for use in in vitro bioassays. Protein concentration was determined spectrophotometrically by the method of the Bradford [56], using Bio Rad Protein Assay with Bovine Serum Albumin (BSA) as the standard. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |