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 | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 371 |
| Molecular Weight | 41565.6 |
| Pi | 9.38 |
| Molecular Weight | 41565.6 |
| Disulphides | 2 |
| Full Sequence |
MASMTGGQQMGRGRDLRRLPQLVGVHPPLQGSSHGAAAIGQPSGELRLRGVAPPPPLQNSSKPRSRAPSNLDAYSHPGPGPGPGSNLTSAPVPSTTTRSLTACPEESPLLVGPMLIEFNIPVDLKLIEQQNPKVKLGGRYTPMDCISPHKVAIIILFRNRQEHLKYWLYYLHPILQRQQLDYGIYVINQAGESMFNRAKLLNVGFKEALKDYDYNCFVFSDVDLIPMNDHNTYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLSINGFPNNYWGWGGEDDDIYNRLAFRGMSVSRPNAVIGKCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYMVLEVQRYPLYTKITVDIGTPS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Boeggeman, E. E., Ramakrishnan, B., Qasba, P. K. (2003) Protein Expression and Purification, 30, 219-229 |
| Project Aim | Folding |
| 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 | 3-4h |
| 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 |
| Refolding Buffer | 50 mM TrisHCl, pH 8.0, 10.58 mM NaCl, 0.44 mM KCl, 2.2 mM MgCl2, 2.2 mM CaCl2, 0.5 M guanidine-HCl, 8 mM cysteamine, 4 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 | 8 mM/ 4 mM respectively |
| Refolding Protocol | Inclusion bodies were sulphonated and then dissolved in solubilization buffer to a protein concentration of 1mg/ml, OD600 1.9-2.0. Protein solution was then diluted 10 fold in refolding solution to give a final protein concentration of 100microg/ml. Refolding was allowed to occur for 48h and then solution was dialyzed. |
| Refolding Assay | Far-UV Circular Dichroism |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine,Polyethylene glycol (PEG) |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |