Refolding Record:
| Protein | |
|---|---|
| Protein Name | Histocompatibility leukocyte antigen-Cw4 |
| Abbreviated Name | HLA-CW4 |
| SCOP Family | MHC antigen-recognition domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P30504 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Extracellular domain |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 276 |
| Molecular Weight | 32104.4 |
| Pi | 5.55009 |
| Molecular Weight | 32104.4 |
| Disulphides | 2 |
| Full Sequence |
MGSHSMR YFSTSVSWPG RGEPRFIAVG YVDDTQFVRF DSDAASPRGE PREPWVEQEG PEYWDRETQK YKRQAQADRV
NLRKLRGYYN QSEDGSHTLQ RMFGCDLGPD GRLLRGYNQF AYDGKDYIAL NEDLRSWTAA DTAAQITQRK WEAAREAEQR RAYLEGTCVE WLRRYLENGK ETLQRAEHPK THVTHHPVSD HEATLRCWAL GFYPAEITLT WQWDGEDQTQ DTELVETRPA GDGTFQKWAA VVVPSGEEQR YTCHVQHEGL PEPLTLRWK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Fan QR, Garboczi DN, Winter CC, Wagtmann N, Long EO, Wiley DC (1996) Proc. Natl. Acad. Sci. USA, 93, 7178-7183 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pLM-1 |
| Expression Protocol | Cells were grown at 37degC in LB medium containing 100microg/ml ampicilin, 34microg/ml chloramphenicol, 0.4% glucose. When cells reached logarithmic phase, protein expression was induced with 0.5mM IPTG. Cells were harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | not stated |
| Solubilization Buffer | 8M urea, 25mM Mes pH 6.0, 10mM EDTA, 1mM DTT |
| Refolding Buffer | 100mM TrisHCl pH 8.3, 400mM L-arginineHCL, 2mM EDTA, 5mM reduced glutathione, 0.5mM oxidized glutathione, 0.2mM PMSF |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.3 |
| Refolding Temperature | 10.0 |
| Protein Concentration | |
| Refolding Time | 72-90h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 5mM/0.5mM |
| Refolding Protocol | Harvested cells were resuspended in 50mM TrisHCl pH 8.0, 25% sucrose, 1mM EDTA, and lysed by freezing and thawing in the presence of 1mg/ml lysozyme. Inclusion bodies were collected and washed extensively, then dissolved in solubilization buffer. Solubilized HLA-Cw4 heavy chain (3microM) and beta-2-microglobulin (2microM) inclusion bodies, together with an HLA-Cw4 specific peptide QYDDAVYKL (60microM) were diluted into 1L of refolding buffer. The refolding mixture was incubated at 10degC for 72-90h, then dialyzed against 10L of H2O at 4deg for 24hr. |
| Refolding Assay | Gel filtration chromatography |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.4M |
| Refolding Yield | |
| Purity | |
| Notes | |