Refolding Record:
| Protein | |
|---|---|
| Protein Name | Defensin |
| Abbreviated Name | n/a |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Crassostrea gigas (Pacific oyster) (Crassostrea an |
| UniProt Accession | Q4GWV4_CRAGI |
| 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 | 65 |
| Molecular Weight | 7008.4 |
| Pi | 8.71 |
| Molecular Weight | 7008.4 |
| Disulphides | 4 |
| Full Sequence |
MKVFVLLTLAVLLMVSADMAFAGFGCPGNQLKCNNHCKSISCRAGYCDAATLWLRCTCTDCNGKK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Gueguen Y, Herpin A, Aumelas A, Garnier J, Fievet J, Escoubas JM, Bulet P, Gonzalez M, Lelong C, Favrel P, Bachère E. (2006) Biologycal Chemistry, 281, 313-323 |
| Project Aim | Purification & characterization |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DE3 |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pEt28a |
| Expression Protocol | Recombinant Cg-Def was expressed in E. coli Rosetta (DE3) pLysS cells (Novagen) transformed with the pET-28a/Cg-Def construct. The cells were grown at 37 °C to A600 0.9 in Luria-Bertani (LB) medium (10 g of bacto-Tryptone, 5 g of bacto yeast extract, and 10 g of NaCl) supplemented with 50 µg/ml kanamycin. Expression of fusion proteins was induced with 0.5 mM isopropyl--D-1-thiogalactopyranoside. After growth at 37 °C for 3 h, bacterial cells were harvested by centrifugation and stored at –20 °C. The cells were lysed by resuspending bacteria pellets in 6 M guanidine HCl in 100 mM Tris-HCl, pH 8.1, followed by sonication at 40% amplitude for 2 min using a Vibra cell Sonifier 450 (Branson Ultrasons, Annemasse, France). The lysate was clarified by centrifugation in a Sorvall SA-600 rotor at 10,000 x g for 30 min at 4 °C prior to protein purification. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.9 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M guanidine HCl, 1 M imidazole, 20 mM Tris-HCl (pH 6.4) |
| Refolding Buffer | 100 mM Tris-HCl buffer at pH 8 |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 48 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | proteins were eluted with two column volumes of 6 M guanidine HCl, 1 M imidazole, 20 mM Tris-HCl (pH 6.4), dialyzed against 5% acetic acid (HOAc) in SpectraPor dialysis membranes (Spectrum Laboratories Inc., Rancho Dominguez), and lyophilized. The methionine residue introduced at the Cg-Def N terminus was subjected to CNBr cleavage by dissolving the lyophilized His6 fusion proteins in 50% formic acid, addition of solid CNBr to 10 mg/ml (final concentration), and incubation of the mixtures for 8 h in the darkness at 25 °C. The cleavage reaction was terminated by adding 10 volumes of water, followed by freezing and lyophilization. Then, the cleaved fusion peptide mixture was reduced in 100 mM Tris-HCl buffer at pH 8 in the presence of 100 mM dithiothreitol. The reaction mixture was purified to homogeneity using a C8 reverse-phase high performance liquid chromatography (RP-HPLC), and the fractions of interest were lyophilized. The peptide was resolved with a 0–55% acetonitrile gradient developed over 30 min at a flow rate of 5 ml/min on a UP10C8 column (Interchrom modulocart uptisphere 10 C8, 250 x 10 mm). The refolding of the Cg-Def was performed at room temperature in 100 mM Tris-HCl buffer at pH 8 during 48 h. Then, the refolded Cg-Def was purified to homogeneity using an additional RP-HPLC step using the same column and eluting conditions as mentioned above. Alternatively, the cleaved fusion peptide mixture was directly folded at pH 8.1 in a buffer solution containing 0.1 M NaHCO3 and 3 mM reduced and 0.3 mM oxidized glutathione in the presence of 2 M urea and 25% N,N-dimethylformamide (15). Then, the folded Cg-Def was purified to homogeneity by RP-HPLC using a C8 column, as described above. Peptide purity was controlled by acid-urea PAGE, and the peptide concentration was estimated by amino acid analysis and UV absorption at 280 nm based on the extinction coefficients of the molecule. Molecular masses of the purified peptides were determined using matrix-assisted laser desorption ionization mode mass spectrometry (MALDI-TOF MS) and electrospray ionization mass spectrometry (ESI-MS). |
| Refolding Assay | Antimicrobial assays |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |