Refolding Record:
| Protein | |
|---|---|
| Protein Name | Human Leukocyte Antigen DR2 |
| Abbreviated Name | HLA DR2 |
| SCOP Family | C1 set domains (antibody constant domain-like) |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01903 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | Beta Polypeptide Chain Lacking Transmembrane Domain |
| Chain Length | 198 |
| Molecular Weight | 23043.7 |
| Pi | 6.25 |
| Molecular Weight | 23043.7 |
| Disulphides | 1 |
| Full Sequence |
GDTRPRFLWQPKRECHFFNGTERVRFLDRYFYNQEESVRFDSDVGEYRAVTELGRPDAEYWNSQKDFLEDRRAAVDTYCRHNYGVGESFTVQRRVQPKVTVYPSKTQPLQHHNLLVCSVSGFYPGSIEVRWFLNGQEEKAGMVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSK
|
| 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,Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | W3310/D3 |
| Expression Temp | 37.0 |
| Expression Time | 2h |
| Expression Vector | p27313 |
| Expression Protocol | Vector pET-11a was used to construct the expression vector by modifications to cloning site. Insert |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Osmotic shock |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 25 mM phosphate buffer, pH 7.4, 8 M urea, 20 mM DTT |
| Refolding Buffer | 12.5 mM phosphate buffer, pH 7.4, 4 M urea, 10 mM DTT, 1.0 M NaCI |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.4 |
| Refolding Temperature | 37.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 30 mM |
| Refolding Protocol | Inclusion bodies solubilized in solubilization buffer. Pellet broken up by mild sonication and allowed to sit at 36degC overnight. Preparation then centriguged (100 000g, 1 hr). Supernatant removed and filtered through 2-pm filter. Recombinant chain purified and refolded by ion exchange chromatography. Which was preformed as follows: 100mg of crude solution was loaded onto 50-ml High Q-50 column. The column was washed subsequently with 90% buffer 12.5 mM phosphate buffer, pH 7.4, 4 M urea, 10 mm DTT) for 60 min at a flow rate of 2.5 m/min. A gradient was run at a flow rate of 2.0 min as follows: 60-180 min 10-30% B refolding buffer, 180-200 min 30%-100%B . Fractions (6.5 ml) were collected and analyzed by SDS-PAGE. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |