Refolding Record:
| Protein | |
|---|---|
| Protein Name | T-Cell receptor D10 single chain |
| Abbreviated Name | TCR D10 |
| SCOP Family | V set domains (antibody variable domain-like) |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | Q5R1G1 |
| 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 | 263 |
| Molecular Weight | 28534.4 |
| Pi | 8.79812 |
| Molecular Weight | 28534.4 |
| Disulphides | 1 |
| Full Sequence |
SSLVPRGSAVSQSPRNKVAVTGGKVTLSCNQTNNHNNMYWYRQDTGHGLRLIHYSYGAGSTEKGDIPDGYKASRPSQENFSLILELATPSQTSVYFCASGGQGRAEQFFGPGTRLTVL GSDYKDDDDKRSGGGGSGGGGSGGSGA QQQVRQSPQSLTVWEGETTILNCSYEDSTFDYFPWYRQFPGKSPALLIAISLVSNKKEDGRFTIFFNKREKKLSLHITDSQPGDSATYFCAATGSFNKLTFGAGTRLAVSPY HHHHHH
|
| Notes | UniProt is 98% homologous to V-alpha portion of construct MBP tag cleaved prior to unfolding and refolding Also see SCOP ref.48936 |
| Expression | |
|---|---|
| Report | Khandekar SS, Bettencourt BM, Wyss DF, Naylor JW, Brauer PP, Huestis K, Dwyer DS, Profy AT, Osburne MS, Banerji J, Jones B (1997) J Biol Chem, 272, 32190-32197 |
| Project Aim | Structure-Function |
| Fusion | N-terminal maltose-binding protein tag/C-terminal His tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 27.0 |
| Expression Time | 3h |
| Expression Vector | pPR998 |
| Expression Protocol | Cells were grown in a 5L bioreactor at 27degC in 4xYT medium containing 2.0% glycerol and 50microg/ml ampicillin. Cells were induced at 1mM IPTG at A600=15 and were harvested 3h after induction with a yield of 60g wet cell paste per litre culture. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 15 |
| Cell Disruption Method | Microfluidizer |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M GdnHCl, 50mM TrisHCl pH 8.0, 0.2M NaCl, 10% glycerol |
| Refolding Buffer | 50mM TrisHCl pH 8.0, 0.2M GdnHCl, 20% glycerol, 0.5M NaCl |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were resuspended in lysis buffer containing 50mM TrisHCl pH 8.0, 0.2M NaCl, 0.1mM 4-(2-aminoethyl)-benzenesulfonyl fluoride-HCl at 10ml/g cells wet weight. Resuspended cells were lysed by passage through a microgluidizer at 15000psi. The supernatant was retained and loaded onto a cross-linked amylose column equilibrated in lysis buffer. The column was washed witn 50mM TrisHCl pH 8.0, 0.2M NaCl, then the protein was eluted with the same buffer containing 1-mM maltose. The purified protein was then denatured with solubilization buffer and applied to a Ni-NTA metal affinity column. After washing with 2 column volumes of solubilization buffer, a refolding gradient was applied from 100% solubilization buffer to 100% refolding buffer. After washing the column with 1 column volume of refolding buffer, the protein was eluted with refolding buffer containing 250mM imidazole. The eluted protein was then further purifiedy by immunoaffinity chromatography and size exclusion. The fusion protein was cleaved by digestion with thrombin and then subjected to further purification steps (Ni-NTA) |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | |
| Purity | |
| Notes | Construct: MPB signal sequence-MBP-beta chain-linker-alpha chain-histage site Maltose binding protein-SSLVPRGS- (beta chain)AVSQSPRNKVAVTGGKVTLSCNQTNNHNNMYWYRQDTGHGLRLIHYSYGAGSTEKGDIPDGYKASRPSQENFSLILELATPSQTSVYFCASGGQGRAEQFFGPGTRLTVL (linker) GSDYKDDDDKRSGGGGSGGGGSGGSGA (alpha chain) QQQVRQSPQSLTVWEGETTILNCSYEDSTFDYFPWYRQFPGKSPALLIAISLVSNKKEDGRFTIFFNKREKKLSLHITDSQPGDSATYFCAATGSFNKLTFGAGTRLAVSPY HHHHHH Parameters without MBP sequence: Number of amino acids: 263 Molecular weight: 28534.4 Theoretical pI: 8.80 |