Refolding Record:
| Protein | |
|---|---|
| Protein Name | Major histocompatibility complex class I |
| Abbreviated Name | MHC-1 |
| SCOP Family | MHC antigen-recognition domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q9TPK7 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 352 |
| Molecular Weight | 38868.5 |
| Pi | 5.41625 |
| Molecular Weight | 38868.5 |
| Disulphides | 2 |
| Full Sequence |
MEPSLLSLFVLGVVALTETRAGSHSLRYFDTAMSRPELGDSQFISVGYVDDQQFVRFDSS
SESPRMEPRAAWMDKVDQEDPNYWEGQTQISRSNAQITRVGLETIRGYYNQSRGGLHTYQ
TMIGCEVHPDGSFRKGFWQHAYDGHDYIALDRETLTWTAADPGAENTKRKWEAERSIAER
YKAYLEEECVQWLKKYLQMGKDVLLRTEPSSARVSRHSGPDGEVSLRCRAQGFYPAEISL
TWLRDGEEQLQDTEFIETRPGGDGTFQKWAAVAMAPGQEDRYSCRVQHEALAQPLSLRWE
PEASSLWVIVGVTAGVLVLVTAVVAGAVILRRRNSGGKGGAYVPGAA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Unpublished (2010) Unpublished, 99, 999 |
| Project Aim | Crystallography |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 4 hrs |
| Expression Vector | pET |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication/French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | (1) 50 mM Tris-HCl pH 8.0, 0.5% (v/v) TritonX-100, 100 mM NaCl, 1 mM NaEDTA (2) same as 1, but no salt or detergent |
| Solubilization Buffer | 20 mM Tris-HCl pH 8.0, 8 M urea, 0.5 mM NaEDTA, 0.1 mM DTT |
| Refolding Buffer | 100 mM Tris-HCL pH 8.0, 2 mM NaEDTA pH 8.0, 400 mM L-Arginine-HCl, 0.5 mM oxidised glutathione, 5 mM reduced glutathione |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | In refold buffer: Pipette in peptide (15 mg/150ul in DMSO), then syringe B2M (12 mg) into the vortex of the buffer (1 injection only of both of these). Then add 1st injection of heavy chain (15 mg). Leave for 12 hours, until next injection (another 15 mg), repeat again 12 hours later (another 15 mg). Dialyse in two changes in dialysis buffer (10 mM Tris pH 8.0) at 4 degrees. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 1% |
| Purity | |
| Notes | |