Refolding Record:
| Protein | |
|---|---|
| Protein Name | Monoclonal Antibody K1 |
| Abbreviated Name | K1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | n/a |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | VH and VL connected as VH-linker-VL |
| Chain Length | 248 |
| Molecular Weight | 26372.5 |
| Pi | 9.33 |
| Molecular Weight | 26372.5 |
| Disulphides | 0 |
| Full Sequence |
MASQVKLKQSGGGLVQPGGSLKVSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTIVYTPSLKDKFIMSRDNAKNTLYLQTSKVRSADTALYYCARRGSHYYGYRTGYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDVVLTQSPAIMSASPGEKVTMTVSASSSVSYMYWHQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGGTKLEIKR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Brinkmann, U., Webber, K., Di Carlo, A., Beers, R., Chowdhury, P., Chang, K., Chaudhary, V,. Gallo, M., Pastan, I. (1997) Int J Cancer, 71, 638-644 |
| Project Aim | Structure-Function |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 90min |
| Expression Vector | pT7-7 |
| Expression Protocol | Cells were grown in superbroth (2% glucose, 0.05% MgSO4, 100microg/ml ampicillin), induced and allowed to incubate. Periplasm was then removed by osmotic shock and sheroplasts were resuspended in Buffer A (50mM Tris, 20mM EDTA, pH 8.0) and incubated (60min, 30degC) with 200microg/ml lysozyme. Triton X-100 and NaCl were added to final concentration of 2% and 0.5 M respectively and the solution was allowed to incubated for a further 30min at 20degC before centrifugation(25,000g, 60min).The resulting pellet was then resuspended in 50mM Tris, 20mM EDTA, pH 8.0 and recentrifuged. This process was repeated twice. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 3 |
| Cell Disruption Method | Osmotic shock |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 0.1 M Tris, pH 8.0, 6 M guanidine, 2 mM EDTA, 0.3 M DTE |
| Refolding Buffer | 0.1 M Tris, 0.5 M L-arginine, 8 mM GSSG, 2 mM EDTA |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 10.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSSG |
| Redox Agent Concentration | 8mM |
| Refolding Protocol | Solubilization buffer was added to dissolve isolated inclusion bodies. The resulting suspension was centrifuged (30,000g, 30min, Room Temp) to remove insoluble material. Refolding was initiated by rapid 100 fold dilution in refolding buffer, this solution was then allowed to incubate. |
| Refolding Assay | Ligand Binding,Sequence Analysis |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.5M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |