Refolding Record:
| Protein | |
|---|---|
| Protein Name | Class I histocompatibility antigen HLA-A |
| Abbreviated Name | HLA-A2 |
| SCOP Family | MHC antigen-recognition domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01892 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | aa.1-271 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 271 |
| Molecular Weight | 31223.5 |
| Pi | 6.0 |
| Molecular Weight | 31223.5 |
| Disulphides | 2 |
| Full Sequence |
GSHSMR YFFTSVSRPG RGEPRFIAVG YVDDTQFVRF DSDAASQRME PRAPWIEQEG PEYWDGETRK VKAHSQTHRV DLGTLRGYYN QSEAGSHTVQ RMYGCDVGSD WRFLRGYHQY AYDGKDYIAL KEDLRSWTAA DMAAQTTKHK WEAAHVAEQL RAYLEGTCVE WLRRYLENGK ETLQRTDAPK THMTHHAVSD HEATLRCWAL SFYPAEITLT WQRDGEDQTQ DTELVETRPA GDGTFQKWAA VVVPSGQEQR YTCHVQHEGL PKPLT
|
| Notes | Heavy chain a.a.1-271 expressed and refolded |
| Expression | |
|---|---|
| Report | Garboczi DN, Hung DT, Wiley CD (1992) Proc. Natl. Acad. Sci. USA, 89, 3429-3433 |
| Project Aim | Crystallography |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | XA90 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pHN1+ |
| Expression Protocol | 400ml of LB medium was inoculated with 100ml stationary phase culture and grown at 37degC. After 30min incubation, 0.5mM IPTG was added and the cells were incubated a further 4h. Cells were collected by centrifugation and the inclusion bodies were isolated. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution with complex partner subunits |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M urea, 50mM 2-(N-morpholino)ethansulfonic acid pH 6.5, 0.1mM EDTA, 0.1mM DTT |
| Refolding Buffer | 100mM TrisHCl pH 8, 400mM L-arginineHCL, 2mM EDTA, 5mM reduced glutathione, 0.5mM oxidized glutathione, 0.5mM PMSF |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 10.0 |
| Protein Concentration | 1microM |
| Refolding Time | 24-36h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a,5mM/0.5mM,n/a |
| Refolding Protocol | The inclusion body pellet was dissolved in 10ml solubilization buffer and then centrifuged (150000g). Refolding was initiated by dilution with denatured beta-2-microglobulin and an antigenic peptide into 200ml refolding buffer. Final protein concentrations were 1microM HLA-A2, 2microM beta-2-microglobulin, 10microM peptide. The refolding mixture was incubated at 10degC for 24-36h. The refolding mixture was concentrated and then subjected to gel filtration HPLC |
| Refolding Assay | HPLC,Crystallography,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 400mM |
| Refolding Yield | 10-15% |
| Purity | |
| Notes | |