Refolding Record:
| Protein | |
|---|---|
| Protein Name | 2-amino-3-ketobutyrateCoA ligase |
| Abbreviated Name | KBL |
| SCOP Family | aminotransferases |
| Structure Notes | |
| Organism | Thermococcus kodakarensis KOD1 |
| UniProt Accession | Unknown |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 395 |
| Molecular Weight | 43971.5 |
| Pi | 5.53043 |
| Molecular Weight | 43971.5 |
| Disulphides | Unknown |
| Full Sequence |
MGKLDWIREELQELKDKGLYVTIRKLESAQGPWVVVDGKKVLNMCSNNYLGLAAHPEIRYAAIRAILDYG
VGAGAVRTIAGTMELHVELEEKLAKFKKREAAILFQSGYNANLGAISALLKKGEDGVFISEELNHASIID
GMRLSGAPKVIYKHIDMEDLKKRLEENKDKKKKIIVSDGVFSMDGDLAPLPEMAELAEQYDAILYIDDAH
GEGVLGDSGRGIVDHFKLHDKVDFEMGTLSKAFGVIGGYVAGPEEAIEYLRQRARPFLFSSAPNPPDVAA
AIAAVEILQRSDELVRKLWDNTNFLQKGLRDLGYDLGNTKHPITPVMLYDEKLAQEFSRRLYDEYNIFAQ
AIVYPTVPLGTARIRLEPSAAHSKEDLQYVIDAFEDLGKKTGFLK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Unpublished (0) J Virol Methods, 0, 0 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | E.coli |
| Expression Strain | BL21c |
| Expression Temp | 37.0 |
| Expression Time | 4hours |
| Expression Vector | PEt21a |
| Expression Protocol | After approximately 4hour at 6500rpm. Pellet was resuspended in 50mM tris buffer pH=7.5 and sonicated.After sonication lysate centrifuged at 9500rpm. KBl appeare as inclusion bodies. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.4 = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Heating in boiling water for 5minut |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mm tris-HCl pH=7.5 |
| Solubilization Buffer | 50mm tris-HCl pH=7.5 |
| Refolding Buffer | 6M urea in tris buffer |
| Pre-Refolding Purification | Heating in boiling water for 5minut |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 2mg/ml |
| Refolding Time | 24hour |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 50mM |
| Refolding Protocol | inclusion bodies were solublized in 6M urea and dialized step wise till urea was completely replaced by 50mM tris-HCl buffer. |
| Refolding Assay | Stability Determination |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 0% |
| Purity | 90% |
| Notes | protein was solublized but not properly folded and pyridoxal phosphate(cofactor) was bounded. |