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 | n/a |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 245 |
| Molecular Weight | 25960.2 |
| Pi | 9.25 |
| Molecular Weight | 25960.2 |
| Disulphides | Unknown |
| Full Sequence |
nagvtqtpkfrvlktgqsmtllcaqdmdheymywyrqdpgmglrlihysvgegttakgevpdgynvsrlkkqnpllglesaapsqtsvyfcasrtatqpqhfgdgtrlsilpggggsggggsggggsggggsgaqqqvkqspqslivqkggisiincayentafdkfpwyqqfpgkgpalliairpdvsekkegrftisfnksakqfslhimdsqpgdsatyfcaasfsgntplvfgkgtrlsvi
|
| Notes | Single chain T-cell receptor, specific for hapten fluorescein in the context of major histocompatibility complex class II. Comprises one V-alpha and one V-beta domain joined via a flexible peptide linker |
| Expression | |
|---|---|
| Report | Novotny J, Ganju RK, Smiley ST, Hussey RE, Luther MA, Recny MA, Siliciano RF, Reinherz EL (1991) Proc. Natl. Acad. Sci. USA, 88, 8646-8650 |
| Project Aim | Structure-Function |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | XL1-Blue |
| Expression Temp | 37.0 |
| Expression Time | 7h |
| Expression Vector | pSS1 |
| Expression Protocol | A 165ml overnight culture grown in LB broth at 37degC with kanamycin (30microg/ml), ampicillin (50mi |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | HPLC |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 10mM TrisHCl pH 8.5, 0.5% NP-40, 1mM PMSF, 1mM EDTA |
| Solubilization Buffer | 20mM TrisHCl pH 8.0, 50mM DTT, 1mM PMSF, 8M urea |
| Refolding Buffer | 1: 20mM sodium acetate pH 5.6, 5mM GSH, 0.5mM GSSG, 2:10mM sodium acetate pH 5.6 |
| Pre-Refolding Purification | HPLC |
| Tag Cleaved | no tag |
| Refolding pH | 5.6 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 3h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 5mM/0.5mM |
| Refolding Protocol | The cell pellet was resuspended in 50mM TrisHCl pH 8.5, 5mM EDTA, 0.3mg/ml lysozyme, 1mM PMSF and incubated on ice for 2h. Nonidet P-40 was added to 0.75% and NaCl was added to 0.35M. The suspension was sonicated and centrifuged (1000g, 20min). The pellet was resuspended in wash buffer containing high salt concentration (1.0M NaCl) and repelleted, then washed twice with low salt wash buffer (no NaCl). The pellet was then resuspended in 5ml solubilization buffer, centrifuged (15min, 1000g) and the supernatant was collected. The protein was then purified by HPLC, collected fracions were combined and resuspended in 8M urea, 10mM TrisHCl pH 8.0, 20mM DTT. The protein was then refolded by rapid dilution 1:100 into refolding buffer 1. After 3h, the solution was dialyzed against refolding buffer 2. 3-{(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate was added to 0.1% and the protein was then concentrated. |
| Refolding Assay | HPLC,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 5-10mg/L cultur |
| Purity | 95% |
| Notes | n/a |