Refolding Record:
| Protein | |
|---|---|
| Protein Name | Capsid protein VP60 |
| Abbreviated Name | VP60 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Rabbit hemorrhagic disease virus (RHDV) |
| UniProt Accession | Q80BA9 |
| 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 | 580 |
| Molecular Weight | 60342.4 |
| Pi | 5.04 |
| Molecular Weight | 60342.4 |
| Disulphides | Unknown |
| Full Sequence |
MEGKARTAPQGEAAGTATTASVPGTTTDGMDPGVVAATSVVTAENSSASVATAGIGGPPQQVDQQETWRTNFYYNDVFTWSVADAPGSILYTVQHSPQNNPFTAVLSQMYAGWAGGMQFRFIVAGSGVFGGRLVAAVIPPGIEIGPGLEVRQFPHVVIDARSLEPVTITMPDLRPNMYHPTGDPGLVPTLVLSVYNNLINPFGGSTNAIQVTVETRPSDDFEFVMIRAPSSKTVDSISPAGLLTTPVLTGVGNDNRWNGQIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFADIDHRRGSASYSGNNATNVLQFWYANAGSAIDNPISKVAPDGFPDMSFVPFNSPNIPAAGWVGFGGIWNSNNGAPAATTVQAYELGFATGAPNNLQPTTNTSGAQTVAKSIYAVVTGTNQNPTGLFVMASGVISTPNANAVTYTPQPDRIVTTPGTPAAAPVGKNSPIMFASVVRRTGDVNAAAGSTNGTQYGTGSQPLPVTIGLSLNNYSSALMPGQFFVWQLTFASGFMEIGLSVDGYFYAGTGA
STTLIDLTELIDVRPVGPRPSKSTLVFNLGGTANGFSYV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Farnós O, Boué O, Parra F, Martín-Alonso JM, Valdés O, Joglar M, Navea L, Naranjo P, Lleonart R. (2005) J Biotechnology, 117, 215-224 |
| Project Aim | Vaccine studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Pichia pastoris |
| Expression Strain | MP36 |
| Expression Temp | 28.0 |
| Expression Time | 72 h |
| Expression Vector | pPSVP60 |
| Expression Protocol | The pPSVP60 expression vector was SalI and PvuII digested to obtain an expression cassette that was used to transform P. pastoris MP36 strain. The electroporation was carried out by the standard procedure described (Becker and Guarente, 1991) in 0.2 cm electroporation cuvettes at 1500 V, 25 μF, 200 Ω using a BioRad Gene Pulser with Pulse Controller (BioRad, USA). His+ transformants were selected in Yeast Nitrogen Base (YNB) minimal medium (Difco Laboratories, USA) supplemented with 2% glucose. Genomic DNA from yeast cells was purified (Rothstein, 1985). Transformant genotypes were analyzed by Southern blot by hybridization with a 32P-labeled AOX1 promoter probe (Southern, 1975 and Sambrook et al., 1989). To determine VP60 protein expression, 11 positive transformants were selected and grown in shake-flasks with 50 mL of YP medium (10% yeast extract, 20% peptone) supplemented with 2% glycerol at 28 °C until an optical density (OD)600 = 4. Cells were recovered, resuspended in the same medium and incubated at 28 °C for 72 h with 0.5% methanol pulses supplied every 12 h. After growth, cells were recovered by centrifugation and disrupted by vortexing with glass beads 0.5–0.75 mm diameter in disruption buffer (50 mM phosphate buffer, pH 7, containing 0.3 M NaCl) with intermittent cooling on ice. Samples from culture supernatant, disruption supernatant and disruption pellet were analyzed by Western blot. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD 4 = 600 |
| Cell Disruption Method | Glass Beads |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM phosphate buffer, pH 7, containing 500 mM NaC |
| Solubilization Buffer | 50 mM phosphate buffer, pH 7, containing 8 M urea and 5 mM EDTA |
| Refolding Buffer | Na2HPO4/NaOH, pH 10 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 10.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The method to purify the recombinant VP60 protein was essentially as described for proteins of veterinary use previously expressed by our group in P. pastoris, which were obtained associated with the cell debris fraction of the transformed yeast (Rodríguez et al., 1994, Boué et al., 1997, Boué et al., 2004, Canales et al., 1997 and García-García et al., 2000). Briefly, yeast cells collected from the bioreactor were centrifuged at 10,600 × g. The pellet was resuspended at 350–450 g L−1 (wet weight) in disruption buffer and disrupted in a glass bead Dynomill KDL disintegrator (Willy A. Bechofen Maschinenfabrik, Basel, Switzerland) equipped with a cold water cooling jacket at a flow rate of 20 mL min−1. The cellular debris was washed once at room temperature with 50 mM phosphate buffer, pH 7, containing 500 mM NaCl, centrifuged at 10,600 × g for 20 min and subjected to another two washings with 50 mM phosphate buffer, pH 7, containing 500 mM NaCl and 1 M urea. Proteins were extracted from the washed-cell debris by homogenization with a Polytron (IKA T-50, Germany) in 50 mM phosphate buffer, pH 7, containing 8 M urea and 5 mM EDTA. A separation step was carried out by centrifugation and the VP60 protein was renatured by slow addition of a buffer containing Na2HPO4/NaOH, pH 10. Most of the yeast contaminants were discarded by acid precipitation at pH 5.0. The supernatant with VP60 protein was concentrated and diafiltered against 50 mM phosphate buffer, pH 8, using a membrane of 30 kDa cut-off (AMICON, USA). The VP60 protein solution was sterile filtrated through a membrane of 0.2 μm (Sartorius, Germany). Total protein concentration was determined using the BCA Protein Assay Kit (Pierce Chemical, USA). Purity was estimated by SDS-PAGE densitometric analysis. |
| Refolding Assay | Western Blot,enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |