Refolding Record:
| Protein | |
|---|---|
| Protein Name | T-cell receptor 2B4 single chain Fv |
| Abbreviated Name | 2B4 TCR-scFv |
| SCOP Family | V set domains (antibody variable domain-like) |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | P01734 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 278 |
| Molecular Weight | 30179.6 |
| Pi | 5.71 |
| Molecular Weight | 30179.6 |
| Disulphides | Unknown |
| Full Sequence |
MGDQV EQSPS ALSLH EGTGS ALRCN FTTTT MRAVQ WFQQN RGSLI NLFYL ASGTK ENGRL KSTFN SKESY STLHI RDAQL EDSGT YFCAA LSGTG GNNKL TFGQG TVLSV IPDIQNP EPAVY QLKDG GGGSG GGGSG GGGS NSKVI QTPRY LVKGQ GQKAK MRCIP EKGHP VVFWY QQNKN NEFKF LINFQ NQEVL QQIDM TEKRF SAECP SNSPC SLEIQ SSEAG DSALY LCASS LNWSQ
DTQYF GPGTR LLVL EDLRNVTTVSSEQKLISEEDLN
|
| Notes | V(alpha) chain-linker-V(beta chain)-Cmyc tag Cmyc tag is a 15a.a. residue tag from human myc oncogene |
| Expression | |
|---|---|
| Report | Kurucz I, Jost CR, George AJT, Andrew SM, Segal DM (1993) Proc. Natl. Acad. Sci. USA, 90, 3830-3834 |
| Project Aim | Functional Studies |
| 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 | 3h |
| Expression Vector | pET11d |
| Expression Protocol | no details provided |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M GdnHCl, 0.3M DTT, 2mM EDTA, 0.1M Tris pH 8.0 |
| Refolding Buffer | 0.4M arginineHCl, 0.4mM reduced glutathione, 2mM EDTA, 0.1mM Tris pH 8.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 10.0 |
| Protein Concentration | 20micrograms/ml |
| Refolding Time | 3-4days |
| Redox Agent | GSSG |
| Redox Agent Concentration | 0.4mM,0.4mM |
| Refolding Protocol | Inclusion bodies (0.3-0.5g wet weight) were dissolved in 6-10ml of solubilization buffer and incubated at room temperature for 1-2h. The protein was then dialyzed against 6M GdnHCl (pH 2.0) at 4degC. Mixed disulfide bonds were produced by the addition of an equal volume of 6M GdnHCl, 0.4M oxidised glutathione, 4mM EDTA, 0.2M Tris pH 8.0 to the denatured protein solution. After 2-3h incubation at room temperature the solution was then dialyzed against 6M GdnHCl/0.1M Tris, 2mM EDTA pH 8.0 at 4degC. The protein was then diluted to a final concentration of 20microgram/ml or less in refolding buffer. The mixture was incubated at 10degC for 3-4 days, the protein was then concentrated. |
| Refolding Assay | Immunoassay |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.4M |
| Refolding Yield | 20-30mg/L |
| Purity | |
| Notes | |