Refolding Record:
| Protein | |
|---|---|
| Protein Name | beta-lactamase |
| Abbreviated Name | beta-lactamase |
| SCOP Family | beta-Lactamase/D-ala carboxypeptidase |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | P00811 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Multi-domain proteins (alpha and beta) |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 359 |
| Molecular Weight | 39551.1 |
| Pi | 8.78 |
| Molecular Weight | 39551.1 |
| Disulphides | Unknown |
| Full Sequence |
APQQINDIVHRTITPLIEQQKIPGMAVAVIYQGKPYYFTWGYADIAKKQPVTQQTLFELGSVSKTFTGVLGGDAIARGEIKLSDPTTKYWPELTAKQWNGITLLHLATYTAGGLPLQVPDEVKSSSDLLRFYQNWQPAWAPGTQRLYANSSIGLFGALAVKPSGLSFEQAMQTRVFQPLKLNHTWINVPPAEEKNYAWGYREGKAVHVSPGALDAEAYGVKSTIEDMARWVQSNLKPLDINEKTLQQGIQLAQSRYWQTGDMYQGLGWEMLDWPVNPDSIINGSDNKIALAARPVKAITPPTPAVRASWVHKTGATGGFGSYVAFIPEKELGIVMLANKNYPNPARVDAAWQILNALQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Harrowing SR, Chaudhuri JB. (2003) J Biochem Biophys Methods., 56, 177-188 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JM109 |
| Expression Temp | 37.0 |
| Expression Time | 24 h |
| Expression Vector | pGB1 |
| Expression Protocol | Batch growth of E. coli JM109/pGB1 was carried out by inoculating 7 l of Luria broth and neomycin growth media in a fermenter (Bioflo IV, New Brunswick Scientific, Hatfield, UK) with 350 ml of overnight E. coli culture grown on the same media. The cells were grown at 37 °C until the optical density (600 nm) reached 0.3–0.4, when the cells were induced with 0.1 mM IPTG [21 and 22]. The fermentation was then continued overnight. The cells were harvested after 24 h culture by centrifugation at 7000 rpm, for 10 min at 4 °C (GSA rotor in a Sorvall RC-5B centrifuge). The cell pellet was stored at −20 °C in a small volume of. Thawed cells in 7 ml of 0.05 M phosphate buffer, pH 7 were disrupted at 26,000 psi in a cell disrupter (One Shot, Constant Systems Limited, UK). The disrupted cells were centrifuged at 3000 rpm, for 15 min 4 °C to pellet the inclusion bodies. Each IB pellet was washed with 20 ml 0.05 M phosphate buffer pH 7 and recentrifuged. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.3-0.4 = 600 |
| Cell Disruption Method | Cell disrupter |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | 20 ml 0.05 M phosphate buffer pH 7 |
| Solubilization Buffer | 5 ml of 6 M GdnHCl, 5 mM DTT |
| Refolding Buffer | 1 M urea in 0.05 M phosphate buffer, pH 7. |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Both the gel filtration media (S-300, fractionation range: 104–1.5×106 Da) and the columns (XK 26/40, XK 16/40 and XK 26/20) were obtained from Amersham Pharmacia (UK). The columns were packed according to the manufacturer\'s instructions. Chromatography was carried out using an automated system (Biologic Workstation, Biorad, Herts, UK), with a UV meter (280 nm), a conductivity meter and fraction collector. All the columns were jacketed and were connected to a circulating water bath (Haake, Fischer, UK). The packed columns were calibrated with dextran and acetone (Table 1). Full-size table (<1K) The packed columns were equilibrated with two column volumes of 1 M urea. The denatured protein sample (8 mg/ml total protein) was loaded through a sample loop (1 or 2 ml) which had been previously rinsed with 6 M GdnHCl, 5 mM DTT. To initiate protein refolding, the mobile phase buffer comprised 1 M urea in 0.05 M phosphate buffer, pH 7. The following sample loadings and flow rates were used (as suggested by the manufacturers instructions for the media and column size): XK 26/40, 2.0 ml of denatured protein sample (1.14% of total bed volume, Vt), flow rate range 0.83–4.00 ml/min; XK 16/40, 1.0 ml (1.48% Vt), 0.33–1.00 ml/min; XK 26/20, 1.0 ml (1.82% Vt), 0.83–4.00 ml/min. All of the elution factions were collected and assayed for total protein concentration, β-lactamase activity and protein aggregation. At least three different flow rates were used for each column, and all refolding runs were performed in triplicate. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |