Refolding Record:
| Protein | |
|---|---|
| Protein Name | Oligopeptide binding protein |
| Abbreviated Name | OppA |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Xanthomonas axonopodis pv. citri |
| UniProt Accession | Q8PP32 |
| 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 | 538 |
| Molecular Weight | 59025.1 |
| Pi | 6.36 |
| Molecular Weight | 59025.1 |
| Disulphides | Unknown |
| Full Sequence |
MALAVLALAGCRQQPAASAGAAGTAPRQGGTLIYATDREPTCLDPHVQGDMPQVFVARQYLDSLVSMDARGRIGPWLARSWEVSADGTAYTFHLREDVRFSDGTPFTAEALKANLDHIADPHTQSSTAGGYIRQYRRTEIVDTHTAIVHLDSAYAAFLEVMAQGFLGLQSPAALARSREQNCQQPVGSGPFKVERWDRQNQLVLVRNPEYAWAPPTARHNGPAYLDRVVWKFIAEPSVRFASLQAGEVDVIDTLPPEAHAPARINPELSLLVANRPGNPTNGTLNLTRAPFDDIRVREAFLRSADVEGALQSVFFGEFARAGGPLSPGTPFYSADFEHAADYDPARANRLLDQAGWTRRDGAGYRSKNGRRLSVDVLMRSQTWPAEQTLWEQVQATARGTGFEVRLHLLSDALLQQRQAAWDYDVRVGYWNTNTADVLRIIFGSQFLAPVGGKGGFHQNGAGFADAQFDAVVAQALATQDPQQRAQLYHQAQRIASGQFLQLTTYPQSTRLGIYKTAHGVALEPALQVTTLYDAWVSK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Balan A, Ferreira RC, Ferreira LC. (2008) Genet Mol Res., 7, 117-126 |
| Project Aim | Protein refolding |
| 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 | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pET28a |
| Expression Protocol | Cultures of the recombinant E. coli BL21 (DE3) strain carrying pETOppA were grown aerobically in Erlenmeyer flasks containing LB with 50 µg/mL kanamycin until mid-log phase (OD 600 = 0.7-0.8) before addition of inducer (0.2 mM IPTG). The cultures were induced aerobically (200 rpm) either for 2 or 4 h or statically overnight at different temperatures (23°, 28° and 37°C). Higher expression levels were achieved after evaluating different induction parameters including growth medium, aeration and incubation temperature during the induction period. Cells were collected by centrifugation and stored at -20°C for approximately 16 h before preparation of the cell extracts. Cell pellets from 1-L cultures were suspended in 10 mL 10 mM Tris-Cl, pH 8.0, 100 mM NaCl, 0.5 mM PMSF, and 3 mM imidazole, and incubated with lysozyme (final concentration of 100 µg/mL) for 1 h in an ice bath. Cells were maintained in ice and sonically disrupted after 5 pulses of 15 s in a Branson Digital Sonifier (Model 450) followed by centrifugation at 18,000 rpm for 20 min to obtain the soluble and non-soluble proteins. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 2 volumes of water and 1 volume of buffer B (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 8 M urea) |
| Solubilization Buffer | 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 8 M urea |
| Refolding Buffer | PBS, pH 7.4, 1 M urea, 10% glycerol, 0.005% Tween 20, 0.5 mM PMSF and 5 mM DTT |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no |
| Refolding pH | 7.4 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 8 h |
| Redox Agent | DTT/PMSF |
| Redox Agent Concentration | 5 mM,0.5 mM,5 mM,0.5 mM |
| Refolding Protocol | Purification and refolding of X. citri OppA The recombinant OppA was purified from insoluble fractions after solubilization with buffer B (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 8 M urea) and addition of nickel-charged Sepharose (ProBond, Invitrogen) slurry (1 mL resin per 15 mg total protein), previously washed with 2 volumes of water and 1 volume of buffer B, and incubated for 1 h under mild agitation. The charged resin was transferred to a plastic column and washed with 10 volumes of buffer B with 10 mM imidazole added, followed by washing with 3 volumes of buffer B plus 20 mM imidazole. The bound OppA was serially eluted using buffers with increasing imidazole concentration. After purification, eluted samples were submitted to SDS-PAGE to confirm purity and protein concentration. Refolding of the denatured protein was attempted by both dialysis and dilution methods, but successful results were achieved only with the dilution method in which protein aliquots (approximately 2 mg) diluted with 100-fold volumes of refolding buffer (PBS, pH 7.4, 1 M urea, 10% glycerol, 0.005% Tween 20, 0.5 mM PMSF and 5 mM DTT) were maintained under vigorous agitation at 4oC for 8 h. The total volume was concentrated on Amicon filters centrifuged at 18,000 rpm at 4°C for 20 min, followed by dialysis with 10% glycerol and 0.5 mM PMSF in PBS, pH 7.4, followed by three dialysis steps with 20 mM Tris-Cl, pH 8.0, and 50 mM NaCl. After centrifugation, the soluble fractions were concentrated with Ultrafree MWCO 30,000 centrifugal filters (Amicon Millipore) and analyzed on 12% acrylamide gels stained with Coomassie blue. In all steps, the protein concentration was determined spectrophotometrically using the Edelhoch method (1967). |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol,tween 20 |
| Additives Concentration | 10%,0.005% |
| Refolding Yield | 120 mg/ml |
| Purity | n/a |
| Notes | n/a |