Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lysozyme |
| Abbreviated Name | Lysozyme |
| SCOP Family | C-type Lysozyme |
| Structure Notes | |
| Organism | Manduca sexta (Tobacco hawkmoth) (Tobacco hornworm |
| UniProt Accession | Q26363 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 138 |
| Molecular Weight | 16087.3 |
| Pi | 8.9 |
| Molecular Weight | 16087.3 |
| Disulphides | Unknown |
| Full Sequence |
MYKLVIFALFAFAYHSEAKHFSRCELVHELRRQGFPENLMRDWVCLVENESSRYTDKVGRVNKNGSRDYGLFQINDKYWCSNGSTPGKDCNVKCSDLLIDDITKASTCAKKIYKRHKFQAWYGWRNHCQGSLPDISSC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | García-Orozco KD, López-Zavala AA, Puentes-Camacho D, Calderón-de-la-Barca AM, Sotelo-Mundo RR. (2005) Biotechnol Lett, 27, 1075-80 |
| Project Aim | Over expression & Renaturation |
| 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 | 18 h |
| Expression Vector | pET11a |
| Expression Protocol | The PCR product and pET11a vector (Novagen) were digested with both enzymes and ligated to produce the recombinant clone, which was sequenced on both strands and transformed into E. coli BL21(DE3) competent bacteria (Novagen). Transformed bacteria were grown in Luria-Bertani medium supplemented with 100 lg ampicillin ml)1 at 37 °C and until the OD at 620 nm was 0.6. Expression was induced by addition of IPTG at 0.4 mM and the cells were grown at the same conditions for 18 h before harvesting by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 620 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 0.05 mM Tris/HCl buffer pH 7.0, 5 mM EDTA, 2 M urea, 5 mM DTT, and 2% Triton X-100 |
| Solubilization Buffer | Tris/HCl pH 7.0, 5 mM EDTA, 8 M guanidine HCl, 5 mM DTT |
| Refolding Buffer | 55 mM Tris/HCl pH 8.5, 1 mM EDTA, 0.1 mM oxidized glutathione, 1 mM reduced glutathione, 264 mM NaCl, 11 mM KCl and 550 mM guanidine HCl |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1/0.1 mM |
| Refolding Protocol | The bacterial pellet was washed with 0.9% NaCl (w/v), and lysed by sonication (four pulses of 10 s at 0 °C) in lysis buffer (0.1 M Tris/HCl pH 8.0, 1 mM EDTA, 5 mM benzamidine, 5 mM dithiotriethol (DTT) and 1 mM phenylmethylsul-fonyl fluoride). The lysate was clarified by centrifugation at 10 000 g for 1 h at 4 °C. Inclusion bodies were cleaned twice using 4 ml g)1 pellet with a wash buffer (0.05 mM Tris/HCl buffer pH 7.0, 5 mM EDTA, 2 M urea, 5 mM DTT, and 2% Triton X-100). To dissolve the pellet, the solution was sonicated for 10 s in ice bath and centrifuged at 22 000 g for 30 min, discarding the supernatant. To remove detergents, the pellet was cleaned twice in the wash buffer but without Triton X-100. The pellet containing the inclusion bodies was dissolved in extraction buffer (0.05 M Tris/HCl pH 7.0, 5 mM EDTA, 8 M guanidine HCl, 5 mM DTT) at 4 ml g)1. Denatured protein was diluted with an equal volume of 0.05 M Tris/HCl pH 7.0, 5 mM EDTA, 5 mM DTT. The solution was centrifuged at 10 000 g for 10 min and the precipitate was discarded. Ms-lyz was refolded by exhaustive dialysis of denatured inclusion bodies against a buffer containing 55 mM Tris/HCl pH 8.5, 1 mM EDTA, 0.1 mM oxidized glutathione, 1 mM reduced glutathione, 264 mM NaCl, 11 mM KCl and 550 mM guanidine HCl (Armstrong et al. 1999) with three exchanges of buffer. The refolded protein was centrifuged at 10 000 g for 1 h. The pellet was discarded and the supernatant was dialyzed exhaustively against phosphate buffer 0.05 M, pH 6.0. |
| Refolding Assay | Protein activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | The refolding temperature not stated |