Refolding Record:
| Protein | |
|---|---|
| Protein Name | Transforming growth factor beta 2 |
| Abbreviated Name | TGFbeta2 |
| SCOP Family | Transforming growth factor (TGF)-beta |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P61812 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 113 |
| Molecular Weight | 12719.6 |
| Pi | 7.68 |
| Molecular Weight | 12719.6 |
| Disulphides | 5 |
| Full Sequence |
ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Han B, Hall FL, Nimni ME. (1997) Protein Expression and Purification, 11, 169-178 |
| 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 | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 5 h |
| Expression Vector | pET |
| Expression Protocol | TGF- B2 expression. For recombinant protein expression, the pET – TGF-B2 – F1 and pET – TGF-B2 – F2 constructs were transformed into the BL21(DE3) strain of E. coli. Primary cultures were grown from single plated colonies and flasks containing 500-ml cultures were inoculated with primary cultures at a 1:50 diluTransformed bacteria were cultured in 2YT medium supplemented with kanamycin (100 ug/ml) until the A 600 reading reached 0.7 – 1.0, after which protein expression was induced in the presence of 0.4 mM IPTG for 5 h at 37 C. Bacterial pellets were collected by centrifugation at 5000g for 10 min and stored at 070C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.7-1.0 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Ni-NTA column |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | Buffer A (20 mM Tris – HCl, 250 mM NaCl, 0.05% NP-40, pH 8.0) |
| Solubilization Buffer | 6 M guanidine – HCl, 0.1 M phosphate buffer, pH 8.0 |
| Refolding Buffer | 8 M urea, pH 8.0 A redox buffer (buffer A with 0.2 mM GSSG/2.0 mM GSH) |
| Pre-Refolding Purification | Ni-NTA column |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a,2/0.2 mM |
| Refolding Protocol | TGF-b2 refolding. After Nickel chelate affinity purification of the His-tagged protein, samples were diluted to appropriate concentrations (10 – 100 mg/ml) in 8 M urea, pH 8.0. A redox buffer (buffer A with 0.2 mM GSSG/2.0 mM GSH was formulated (see Results), chilled on ice, and slowly added to the purified TGF B2 fusion proteins to dilute the urea to 1.3 M (6X dilution) and initiate the oxidative refolding. After vigorous mixing, the recombinant TGF-b2 fusion proteins were allowed to anneal into a thermodynamically stable structure at 4􏰈C for various time intervals under specified conditions. After prolonged oxidative refolding, samples were dialyzed either against buffer B (500 mM NaCl, 20 mM Tris – HCl, 10% glycerol, pH 8.0) or 10% acetonitrile with 0.05% TFA. Samples in acetonitrile were then either frozen directly or lyophilized and reconstituted into 1% BSA/40 mM HCl prior to freezing. |
| Refolding Assay | Unspecified,Binding assay,Biological assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | |