Refolding Record:
| Protein | |
|---|---|
| Protein Name | T-cell receptor |
| Abbreviated Name | TCR |
| SCOP Family | V set domains (antibody variable domain-like); C1 set domains (antibody constant domain-like) |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P04435 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 112 |
| Molecular Weight | 12794.2 |
| Pi | 8.73 |
| Molecular Weight | 12794.2 |
| Disulphides | 1 |
| Full Sequence |
GVSQNPRHNITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLAGLNQPQHFGDGTRLSIL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | De Marzí MC, Fernández MM, Sundberg EJ, Molinero L, Zwirner NW, Llera AS, Mariuzza RA, Malchiodi EL. (2004) FEBS Letters, 271, 4075-4083 |
| Project Aim | Cloning and expression |
| Fusion | None |
| 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 | 3-5 h |
| Expression Vector | pET17b |
| Expression Protocol | Luria–Bertani broth (LB) agar plates containing 50 ug·mL−1 of kanamycin or 100 ug·mL−1 of ampicillin were incubated overnight at 37 °C from transforming BL21(DE3) glycerol stocks. One litre of LB medium was inoculated with 10 mL overnight culture and grown with shaking at 37 °C to an attenuance of 0.8 at 600 nm. TCR expression was induced with 1 mm isopropyl thio-B-d-galactoside for 3–5 h. Cells were harvested by centrifugation at 2100 g for 20 min. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.8 = 600 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 0.5% (v/v) Triton X-100 and 100 mm NaCl,50 mm Tris/HCl, pH 7.5, 1 mm EDTA, pH 8, and 1 mm dithiothreitol (DTT) |
| Solubilization Buffer | 8 m urea, 100 mm Tris/HCl, pH 7.5, 10 mm EDTA and 1 mm DTT |
| Refolding Buffer | 1 m arginine/HCl, pH 7.5, 2 mm EDTA, 100 mm Tris/HCl, pH 7.5, 6.3 mm cysteamine, 3.7 mm cystamine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 48 h |
| Redox Agent | cysteamine/cystamine |
| Redox Agent Concentration | 1 mM,6.3/3.7 mm |
| Refolding Protocol | The bacterial pellet of hVB5.2mCB15 was resuspended in lysis buffer [50 mm Tris/HCl, pH 7.5, 1 mm EDTA, pH 8, and 1 mm dithiothreitol (DTT)] and passed through a French press twice at 1300 psi. The lysate was centrifuged at 7700 g for 15 min and the pelleted inclusion bodies were washed four times with 0.5% (v/v) Triton X-100 and 100 mm NaCl in lysis buffer. The inclusion bodies were then washed with 2 m urea in 2 m NaCl, 50 mm Tris/HCl, pH 7.5, 1 mm DTT, with 4 m urea in the same buffer, and finally with 100 mm Tris/HCl, pH 7.5, 1 mm EDTA and 1 mm DTT. Inclusion bodies were then solubilized in 8 m urea, 100 mm Tris/HCl, pH 7.5, 10 mm EDTA and 1 mm DTT. Concentration of solubilized inclusion bodies was estimated in a Coommassie Blue stained SDS/PAGE, using different concentrations of BSA and then diluted 1 : 5 in 6 m guanidine, 10 mm acetate buffer, pH 4.2, and 10 mm EDTA. Denatured B chain was added dropwise to the renaturation buffer (1 m arginine/HCl, pH 7.5, 2 mm EDTA, 100 mm Tris/HCl, pH 7.5, 6.3 mm cysteamine, 3.7 mm cystamine) under vigorous stirring to a final concentration of 20–50 Ug·mL−1 during 48 h at 4 °C. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |