Refolding Record:
| Protein | |
|---|---|
| Protein Name | Haemoglobin alpha subunit |
| Abbreviated Name | Hb |
| SCOP Family | Globins |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P69905 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 141 |
| Molecular Weight | 15126.4 |
| Pi | 8.73104 |
| Molecular Weight | 15126.4 |
| Disulphides | 0 |
| Full Sequence |
VLSPADKTNV KAAWGKVGAH AGEYGAEALE RMFLSFPTTK TYFPHFDLSH GSAQVKGHGK KVADALTNAV AHVDDMPNAL SALSDLHAHK LRVDPVNFKL LSHCLLVTLA AHLPAEFTPA VHASLDKFLA SVSTVLTSKY R
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Tame J, Shih DT, Pagnier J, Fermi G, Nagai K (1991) J Mol Biol, 218, 761-767 |
| Project Aim | Structure-Function |
| Fusion | N-terminus CII and beta-globin residues 1-18 |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | QY13 |
| Expression Temp | 42.0 |
| Expression Time | 3-4h |
| Expression Vector | pLmp10 |
| Expression Protocol | Cells were grown in 2TY medium at 30degC until A600 reached 1.0. The temperature was then raised to 42degC and the culture incubated a further 3-4h before harvesting cells. |
| Method of Induction | Temperature Shift |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | not stated |
| Solubilization Buffer | 6M GdnHCl, 50mM TrisHCl pH 8.0, 1mM EDTA, 1mM DTT |
| Refolding Buffer | 20mM potassium acetate pH 5.7, 1mM EDTA |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Tag Cleaved | yes |
| Refolding pH | 5.7 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.25mg/ml |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The inclusion bodies containing fusion protein were washed, then dissolved in solubilization buffer using a homogenizer. The protein was then dialysed against water, and then 8M urea, 25mM Tris acetate (pH 5.0), 1mM EDTA, 1mM DTT. The fusion protein was then purified using a CM-Sepharose column and a Sephacryl S-200 column, the fusion protein was then cleaved with Factor Xa. The released alpha-globin was then dialysed against 8M urea, 25mM Tris acetate pH 5, 1mM EDTA, 1mM DTT and diluted to a concentration of 5mg/ml with the same buffer. The protein was then diluted to a final concentration of 0.25mg/ml in ice cold refolding buffer. Then hemin dicyanide and beta-globin were added to a 1:2 molar excess. The reconstituted haemoglobin tetramers were then reduced and purified. |
| Refolding Assay | Crystallography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |