Refolding Record:
| Protein | |
|---|---|
| Protein Name | Penicillin-binding protein 1 |
| Abbreviated Name | PBP1 |
| SCOP Family | Penicillin binding protein dimerisation domain |
| Structure Notes | |
| Organism | Mycobacterium tuberculosis |
| UniProt Accession | Q8VKS5 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | (V2-Q94)PBP1 |
| Chain Length | 599 |
| Molecular Weight | 62559.3 |
| Pi | 5.11 |
| Molecular Weight | 62559.3 |
| Disulphides | 0 |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSHMGGSTITQQYVKNALVGSAQHGWSGLMRKAKELVIATKMSGEWSKDDVLQAYLNIIYFGRGAYGISAASKAYFDKPVEQLTVAEGALLAALIRRPSTLDPAVDPEGAHARWNWVLDGMVETKALSPNDRAAQVFPETVPPDLARAENQTKGPNGLIERQVTRELLELFNIDEQTLNTQGLVVTTTIDPQAQRAAEKAVAKYLDGQDPDMRAAVVSIDPHNGAVRAYYGGDNANGFDFAQAGLQTGSSFKVFALVAALEQGIGLGYQVDSSPLTVDGIKITNVEGEGCGTCNIAEALKMSLNTSYYRLMLKLNGGPQAVADAAHQAGIASSFPGVAHTLSEDGKGGPPNNGIVLGQYQTRVIDMASAYATLAASGIYHPPHFVQKVVSANGQVLFDASTADNTGDQRIPKAVADNVTAAMEPIAGYSRGHNLAGGRDSAAKTGTTQFGDTTANKDAWMVGYTPSLSTAVWVGTVKGDEPLVTASGAAIYGSGLPSDIWKATMDGALKGTSNETFPKPTEVGGYAGVPPPPPPSEVPPSETVIQPTVEIAPGITIPIGPPTTITLAPPPPAPPAATPTPPP
|
| Notes | Mutant lacking residues V2-Q94 |
| Expression | |
|---|---|
| Report | Bhakta, S., Basu, J. (2002) Biochem J., 361, 635-639 |
| Project Aim | Functional Studies,Recombinant Protein Expression |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 30.0 |
| Expression Time | 3h |
| Expression Vector | pET28a |
| Expression Protocol | Cells were grown in LB broth (supplemented with 50microg/ml kanamycin), at 37degC, overnight with shaking. Cells were then induced and incubated prior to harvesting. Harvested cells were resusupended in Buffer A (10mM TrisHCl, pH 7.4, 1 mM MgCl2, 1 microg/ml DNAse), and sonicated (200W for 2 min). Lysate was then centrifuged(600g, 10min), and inclusion bodies isolated by further centrifugation (5000g, 10min). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 5 M guanidineHGl, 150 mM TrisHCl, pH 8.0, 1 mM DTT, 1 mM EDTA |
| Refolding Buffer | 400 mM TrisHCl, pH 8.0, 20% glycerol, 1 mM EDTA |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 20.0 |
| Protein Concentration | n/a |
| Refolding Time | 16h |
| Redox Agent | DTT |
| Redox Agent Concentration | 1 mM |
| Refolding Protocol | Inclusion bodies were solubilized, and underwent dialysis against refolding buffer. Refolded protein was the purified by affinity chromatography on Ni-NTA-agarose as describe previously. |
| Refolding Assay | Ligand Binding,Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |