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 | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 692 |
| Molecular Weight | 72654.8 |
| Pi | 5.54 |
| Molecular Weight | 72654.8 |
| Disulphides | 0 |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSHMVTFTMAYLIVDVPKPGDIRTNQVSTILASDGSEMAKIVPPEGNRVDVNLSQVPMHVRQAVIAAEDRNFYSNPGFSFTGFARAVKNNLFGGDLQGGSTITQQYVKNALVGSAQHGWSGLMRKAKELVIATKMSGEWSKDDVLQAYLNIIYFGRGAYGISAASKAYFDKPVEQLTVAEGALLAALIRRPSTLDPAVDPEGAHARWNWVLDGMVETKALSPNDRAAQVFPETVPPDLARAENQTKGPNGLIERQVTRELLELFNIDEQTLNTQGLVVTTTIDPQAQRAAEKAVAKYLDGQDPDMRAAVVSIDPHNGAVRAYYGGDNANGFDFAQAGLQTGSSFKVFALVAALEQGIGLGYQVDSSPLTVDGIKITNVEGEGCGTCNIAEALKMSLNTSYYRLMLKLNGGPQAVADAAHQAGIASSFPGVAHTLSEDGKGGPPNNGIVLGQYQTRVIDMASAYATLAASGIYHPPHFVQKVVSANGQVLFDASTADNTGDQRIPKAVADNVTAAMEPIAGYSRGHNLAGGRDSAAKTGTTQFGDTTANKDAWMVGYTPSLSTAVWVGTVKGDEPLVTASGAAIYGSGLPSDIWKATMDGALKGTSNETFPKPTEVGGYAGVPPPPPPSEVPPSETVIQPTVEIAPGITIPIGPPTTITLAPPPPAPPAATPTPPP
|
| Notes | n/a |
| 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 full length PBP1, in membranes, was obtained by centrifugation (100,000g, 30min). Recombinant PBP1 was isolated from membrane, by suspension in 5ml of 10mM Tris, HCl containing 1 % Zwittergent 3-12 and maintained at 4degC for 40min. After centrifugation(100,000g,30min) the supernatant was removed and NaCl was added to a final concentration of 0.5M. The supernatant was then loaded onto a Ni2+-NTA agarose column, equilibrated with Buffer B (50mM sodium phosphate, pH 7.4, and 0.5 M NaCl). The column was then washed with Buffer B containing 100mM imidazole and PBP1 was then eluted with a gradient of 100-2000mM imidazole/1% Zwittergent 3-12 in Buffer B. |
| 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 |