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 | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 276 |
| Molecular Weight | 31808.2 |
| Pi | 6.01 |
| Molecular Weight | 31808.2 |
| 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 PKPLTLRWE
|
| Notes | Heavy chain enconding 1-275 of HLA-A2 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 | not stated |
| Expression Vector | pHN1+ |
| Expression Protocol | 1L of cells was incubated at 37degC and induced by the addition of 1mM IPTG. When OD650 reached 1.8-2.0, cells were harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 10mM TrisHCl pH 8 |
| Solubilization Buffer | 100mM TrisHCl pH 8.0m 8M urea |
| Refolding Buffer | 25mM 2-(N-morpholino)ethanesulfonic acid pH 6.5, 150mM NACl |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 6.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | overnight |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Cells were resuspended in 20ml of 10mM TrisHCl pH 8 containing 100microg/ml lysozyme, 50microg/ml PMSF, 20microg/ml DNase, 20microg/ml RNase and 1mM EDTA. Cells were incubated for 20min at 22degC, then lysed by conication and centrifuged (20min, 10000g). The pellet was washed in 20ml wash buffer, then dissolved in 10ml solubilization buffer for 1h at 4degC. The protein was reconsituted by mixing 3microM of the heavy chain protein with 120microM HLA-A2-peptide molecule and 6microM beta-2-microglobulin in refolding buffer containing 6M NaSCN. This mixture was dialysed overnight at 4degC against refolding buffer, then centrifuged (10min, 14000rpm, 4degC). |
| Refolding Assay | HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 10-15% |
| Purity | |
| Notes | |