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 | He XH, Xu LH, Liu Y. (2005) World J Gastroenterol, 11, 4180-4187 |
| Project Aim | Functional Studies |
| Fusion | C-terminal BirA substrate peptide |
| 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 h |
| Expression Vector | pET-3c |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mmol/L Tris-HCl pH8.0, 100 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT and 5 g/L Triton X-100 |
| Solubilization Buffer | 20 mmol/L 2-(N-morpholino)ethanesulfonic acid (MES) (pH 6.0, containing 8 mol/L urea 10 mmol/L EDTA and 0.1 mmol/L DTT |
| Refolding Buffer | 100 mmol/L Tris-HCl pH 8.0, containing 400 mmol/L L-arginine, 2 mmol/L EDTA, 5 mmol/L reduced glutathione, 0.5 mmol/L oxidized glutathione and 0.2 mmol/L PMSF |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 10.0 |
| Protein Concentration | |
| Refolding Time | 3 days |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Briefly, 2 mg of peptide (NLV or GIL peptide) dissolved in DMSO was added dropwisely to 200 mL of pre-chilled refolding buffer (100 mmol/L Tris-HCl pH 8.0, containing 400 mmol/L L-arginine, 2 mmol/L EDTA, 5 mmol/L reduced glutathione, 0.5 mmol/L oxidized glutathione and 0.2 mmol/L PMSF). Then, 6 mg A2-BSP in 1 mL injection buffer containing 3 mol/L guanidine-HCl pH 4.2, 10 mmol/L sodium acetate and 10 mmol/L EDTA was forcefully injected to the stirring reaction through a 26-gauge needle as close to the stir bar as possible. Five micrograms of beta2-microglobulin was injected similarly, and the refolding mixture was incubated at 10 degree C for three days. At the end of the incubation, 200 mL of the refolding mixture was concentrated to 5 mL with an ultrafiltration apparatus (Amicon, Millipore, Bedford, MA, USA) containing a 10 ku molecular mass cutoff membrane, and dialyzed against 10 mmol/L Tris-HCl buffer (pH 8.0, containing 0.2 mmol/L PMSF). The refolded monomeric HLA-A2 was centrifuged to eliminate precipitates. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | approx. 10% |
| Purity | 85% |
| Notes | |