Refolding Record:
| Protein | |
|---|---|
| Protein Name | T cell receptor beta 2.1 chain |
| Abbreviated Name | hVbeta2.1 |
| SCOP Family | V set domains (antibody variable domain-like) |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | n/a |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | C13A |
| Chain Length | 118 |
| Molecular Weight | 12822.5 |
| Pi | 7.01 |
| Molecular Weight | 12822.5 |
| Disulphides | 0 |
| Full Sequence |
GAVVSQHPSRVIAKSGTSVKIECTSLDFQATTMFWYRQFPKQSLMLMATSWEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSALAGSGSSTDTQYFGPGTRLTVL
|
| Notes | A number of alanine scanning mutants for this construct were also made. These include:Q28, T30, R50, I51, D52, H53, T55, Y56, V61, K62, D63, K64, L66, N68, H69, S98, S101. |
| Expression | |
|---|---|
| Report | Buonpane, R. A., Moza, B., Sundberg, E. J., and Kranz, D. M (2005) J Biol Chem, 353, 308-321 |
| 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 | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pT7-7 vector |
| Expression Protocol | Cells were cultured in LB medium containing 100microg/ml ampicillin and 34 microg/ml chloroamphenicol and induced at log phase. After incubation cells were harvested by centrifugation, lysed and inclusion bodies isolated. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 50 mM TrisCl, pH 8.0, 8 M Urea, 10 mM DTT, 0.5 mM EDTA |
| Refolding Buffer | 100 mM TrisCl, pH 8.5, 1 M arginine, 2 mM EDTA, 6.3 mM cysteamine, 3.7 mM cystamine |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 3 days |
| Redox Agent | cysteamine/cystamine |
| Redox Agent Concentration | 6.3 mM/ 3.7 mM respectively |
| Refolding Protocol | Isolated inclusion bodies were extensively washed prior to solubilization. After treatment approximately 50mg of solubilized inclusion body material was diluted in 1 L of refolding buffer and allowed to incubate. |
| Refolding Assay | Surface plasmon resonance binding |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 1 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |