Refolding Record:
| Protein | |
|---|---|
| Protein Name | Human leukocyte antigen DR2 alpha chain |
| Abbreviated Name | HLA DR2 alpha |
| SCOP Family | MHC antigen-recognition domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01903 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 233 |
| Molecular Weight | 25986.8 |
| Pi | 4.96145 |
| Molecular Weight | 25986.8 |
| Disulphides | Unknown |
| Full Sequence |
IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVD
MAKKETVWRLEEFGRFASFEAQGALANIAVDKANLEIMTKRSNYTPITNVPPEVTVLTNS
PVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTTGVSETVFLPREDHLFRKFHYLPFLP
STEDVYDCRVEHWGLDEPLLKHWEFDAPSPLPETTENVVCALGLTVGLVGIIIGTIFIIK
GVRKSNAAERRGPL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Arimilli S, Cardoso C, Mukku P, Baichwal V, Nag B (1995) J Biol Chem, 270, 971-977 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | W3310(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 2 |
| Expression Vector | pET-11a |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 25mM phosphate pH 7.4, 8M urea, 20mM DTT |
| Refolding Buffer | PBS |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.5mg/ml |
| Refolding Time | 18h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were grown in L-broth containing 0.4% glucose, 100microg/ml ampicillin and 15microg/ml tetracycline. Cells were induced in mid-log growth with 0.4mM IPTG then grown for a further 2h. Cells were then harvested. Inclusion bodies were dissolved in solubilization buffer - IBs were pelleted then sonicated and incubated at 37degC overnight. The preparation was then centrifuged (1h x 100000g). The supernatant was filtered then loaded onto a 50ml High Q-50 resin. The column was washed with 90% buffer A (12.5mM phosphate buffer pH 7.4, 4M urea 10mM DTT) for 60min x 2.5ml/min. A gradient was then run at 2.0ml/min as follows: 60-180 min 10-30% Buffer B (12.5mM phosphate pH 7.4, 4M urea, 10mM DTT, 1.0M NaCl), 180-200min 30-100% Buffer B. 6.5ml fractions were collected. The protein was dialysed against PBS at 0.5mg/ml for 18h at 25degC, then mixed with beta-chain and a 1-50 molar excess of myelin basic protein (MBP) peptide in reconstitution buffer (50mM sodium phosphate pH 7.5, 1mM EDTA, 3mM reduced glutathione, 0.3mM oxidized glutathione, 25% (v/v) glycerol, 10mM DTT) to form a complex. The complex was then dialyzed against 4L PBS at 4degC with two changes. The DR2-MBP peptide complex was then purified further by immunoaffinity chromatography using immobilized L243 monoclonal antibody. |
| Refolding Assay | ELISA |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | >95% |
| Notes | This protocol applies to the DR2 alpha chain lacking the transmembrane region See also refolding record for HLA DR-alpha Also see Nag et al., J Biol Chem (1994) 269:10061-10070 for details of refolding and purification |