Refolding Record:
| Protein | |
|---|---|
| Protein Name | MHC ClassII HLA-DR-beta |
| Abbreviated Name | n/a |
| SCOP Family | C1 set domains (antibody constant domain-like) |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q29769 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Tetramer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 114 |
| Molecular Weight | 13489.9 |
| Pi | 5.9 |
| Molecular Weight | 13489.9 |
| Disulphides | Unknown |
| Full Sequence |
SSPLALAGDTRPRFLEYSTGECYFFNGTERVRFLDRYFHNQEEFVRFDSDVGEYRAVTELGRPDAEYWNSQKDFLEDRRALVDTYCRHNYGVGESFTVQRRVHPKVTVYPSKTQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Cunliffe SL, Wyer JR, Sutton JK, Lucas M, Harcourt G, Klenerman P, McMichael AJ, Kelleher AD. (2002) Eur J Immunol, 32, 3366-3375 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | HMS 174(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 00 |
| Expression Vector | pET16B |
| Expression Protocol | E. coli strain HMS 174(DE3) pLysS (Novagen) was transfected by heat shock with plasmids containing either modified - or -chains. DRA1*0101 - and DRB1*0101/DRB1*1101 -chains were expressed separately in E. coli as inclusion bodies after induction by isopropyl -D-thiogalactoside (IPTG) |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 5.8 M guanidine-HCl, 50 mM Tris pH 8.0 |
| Refolding Buffer | 25% glycerol, 40 mM Na2HPO4, 10 mM NaH2PO4, 2 mM EDTA, 5 mM glutathione (reduced form), 0.5 mM glutathione (oxidized form) |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 5-0.5 mM |
| Refolding Protocol | The proteins were purified from inclusion bodies, denatured in Guanidine buffer (5.8 M guanidine-HCl, 50 mM Tris pH 8.0), and then oxidized for 48 h. DR1 without the linked peptide was folded in buffer [25% glycerol, 40 mM Na2HPO4, 10 mM NaH2PO4, 2 mM EDTA, 5 mM glutathione (reduced form), 0.5 mM glutathione (oxidized form)], in the presence of excess peptide [9]. Linked -chains were folded in the same manner but without the addition of free peptide. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 25% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |