Refolding Record:
| Protein | |
|---|---|
| Protein Name | Beta 1, 4 galactosyltransferase I |
| Abbreviated Name | B4(Gal-T1) |
| SCOP Family | beta 1,4 galactosyltransferase (b4GalT1) |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | P08037 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | Mutant D242A |
| Chain Length | 371 |
| Molecular Weight | 41521.6 |
| Pi | 9.45 |
| Molecular Weight | 41521.6 |
| Disulphides | 2 |
| Full Sequence |
MASMTGGQQMGRGRDLRRLPQLVGVHPPLQGSSHGAAAIGQPSGELRLRGVAPPPPLQNSSKPRSRAPSNLDAYSHPGPGPGPGSNLTSAPVPSTTTRSLTACPEESPLLVGPMLIEFNIPVDLKLIEQQNPKVKLGGRYTPMDCISPHKVAIIILFRNRQEHLKYWLYYLHPILQRQQLDYGIYVINQAGESMFNRAKLLNVGFKEALKAYDYNCFVFSDVDLIPMNDHNTYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLSINGFPNNYWGWGGEDDDIYNRLAFRGMSVSRPNAVIGKCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYMVLEVQRYPLYTKITVDIGTPS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Boeggeman, E., Qasba, P. K. (2002) Glycobiology, 12, 395-407 |
| Project Aim | Structural Studies |
| Fusion | N-terminal T7 tag |
| 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 | 4h |
| Expression Vector | pET-23a |
| Expression Protocol | Cells were grown in LB broth containing 50microg/ml ampicillin. Following induction and incubation, cells were harvested by centrifugation (2000g, 20min) and inclusion bodies isolated. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.7 |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | n/a |
| Solubilization Buffer | 5M guanidine-HCl, 0.3 N siduyn sulfite |
| Refolding Buffer | 50 mM TrisHCl, pH 8.0, 5 mM ethylenediamine tetra-acetic acid, 0.5 M guanidine-HCl, 4 mM cysteamine, 2 mM cystamine, 0.055% PEG-4000, 0.55M L-arginine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 48h |
| Refolding Time | n/a |
| Redox Agent | cysteamine/cystamine |
| Redox Agent Concentration | 4 mM/ 2 mM respectively |
| Refolding Protocol | Isolated inclusion bodies were washed with PBS. 100mg of inclusion body was then dissolved in 10ml solubilization buffer, to which 50mM disodium 2-nitro-5-thiosulfobenzoate solution is added to sulfonate the free thiols on the protein. Sulfonated proteins were diluted 10x in water, precipitated and collected by centrifugation(10,000g). Centrifuged proteins were washed 4x with water, before re-dissolving in 5M guanidine HCl, 4 mM cysteamine, 2 mM cystamine. Refolding was initiated by 10x dilution followed by dialysis after a 48h incubation. |
| Refolding Assay | Enzyme activity,Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 3-5mg |
| Purity | n/a |
| Notes | n/a |