Refolding Record:
| Protein | |
|---|---|
| Protein Name | HLA class I histocompatibility antigen B |
| Abbreviated Name | HLA B44 |
| SCOP Family | MHC antigen-recognition domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P30481 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Extracellular domain (aa.1-276) of mature protein |
| Chimera | n/a |
| Variants | B*4402 variant - D116, D156 |
| Chain Length | 276 |
| Molecular Weight | 31938.3 |
| Pi | 5.36 |
| Molecular Weight | 31938.3 |
| Disulphides | 2 |
| Full Sequence |
GSHSMR YFYTAMSRPG RGEPRFITVG YVDDTLFVRF DSDATSPRKE PRAPWIEQEG PEYWDRETQI SKTNTQTYRE NLRTALRYYN QSEAGSHIIQ RMYGCDVGPD GRLLRGYDQD AYDGKDYIAL NEDLSSWTAA DTAAQITQRK WEAARVAEQD RAYLEGLCVE SLRRYLENGK ETLQRADPPK THVTHHPISD HEVTLRCWAL GFYPAEITLT WQRDGEDQTQ DTELVETRPA GDRTFQKWAA VVVPSGEEQR YTCHVQHEGL PKPLTLRWEP
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Macdonald W, Williams DS, Clements CS, Gorman JJ, Kjer-Nielsen L, Brooks AG, McCluskey J, Rossjohn J, Purcell AW (2002) FEBS Letters, 527, 27-32 |
| Project Aim | Crystallography |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(RIL) |
| Expression Temp | 37.0 |
| Expression Time | 12h |
| Expression Vector | pET |
| Expression Protocol | Cells were grown and protein expression was induced with 1mM IPTG when A600 reached 0.6. Cells were grown a further 12h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Chemical |
| Lytic Agent | Detergents |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution with complex partner subunits |
| Wash Buffer | 50mM TriHCl, 1mM NaEDTA, 1mM DTT pH 8.0 |
| Solubilization Buffer | 25mM MES, 8M urea, 10mM NaEDTA pH 6.0 |
| Refolding Buffer | 0.1M Tris, 2mM EDTA, 400mM L-arginineHCl, 0.5mM GSH, 5mM GSSG, pH 8.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 24h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 0.5mM/5mM |
| Refolding Protocol | Bacteria were lysed in 50mM TriHCl pH 8.0, 1% Triton X-100, 1% sodium deoxycholate, 100mM NaCl and 10mM DTT. Inclusion bodies were isolated by centrifugation then washed twice with washing buffer, firstly with 0.5% Triton X-100 and 100mM, and then without. Inclusion bodie were then dissolved in solubilization buffers with 1microg/ml pepstatin A and 200microM PMSF. 60mg of protein was then refolded with 20mg beta-2-microglobulin and 30mg of the cpeptide EEFGRAFS in refolding buffer. Following refolding, the protein was dialyzed overnight against Milli Q and then purified by using a DE52 ion exchange column, followed by a Superdex 75 gel filtration column and then a MonoQ column. |
| Refolding Assay | Crystallography,SDS-PAGE,ELISA |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 400mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |