Refolding Record:
| Protein | |
|---|---|
| Protein Name | Protein A33 |
| Abbreviated Name | A33R |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Vaccinia virus (strain Western Reserve / WR) (VACV |
| UniProt Accession | P68617 |
| 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 | 187 |
| Molecular Weight | 20506.1 |
| Pi | 5.33 |
| Molecular Weight | 20506.1 |
| Disulphides | Unknown |
| Full Sequence |
MMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSAFLIVRLNQCMSANEAAITDAAVAVAAASSTHRKVASSTTQYDHKESCNGLYYQGSCYILHSDYQLFSDAKANCTAESSTLPNKSDVLITWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCVKTMN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Fang M, Cheng H, Dai Z, Bu Z, Sigal LJ. (2006) Virology, 345, 231-43 |
| Project Aim | Vaccine studies |
| 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 | 4 h |
| Expression Vector | pET28a |
| Expression Protocol | The coding sequences for the extraviral domains of ECTV Moscow EVM135 and VACV Western Reserve A33R (Galmiche et al., 1999 and Roper et al., 1996) were amplified by PCR from genomic DNA. To facilitate purification, the recombinant proteins were designed to contain a terminal 6 × His tag. Because VACV A33R and its orthologs such as ECTV EVM135 are type II proteins (Roper et al., 1996), their C-termini are exposed at the surface of EEV. Therefore, the 6 × His tag was fused to the N-terminus. For EVM135, we used 5′AAACCATGGGCCATCACCATCACCATCACTGCATGTCTGCTAACGAGGTTG and 5′ AAACTCGAGTTAGTTCATTATTTTAACACAAAAATACTTTC as the forward and reverse primers respectively. For A33R the forward and reverse primers used were 5′AAACCATGGGCCATCACCATCACCATCACTGCATGTCTGCTAACGAGGCTG and AAACTCGAGTTAGTTCATTGTTTTAACACAAAAATACTTTC respectively. The amplified products were cloned into pET-28a(+) vector and transformed into DH5 competent cells. The expression vector was verified by DNA sequencing and then transformed into BL21(DE3) competent cells for expression. The transformed BL21(DE3) cells were grown overnight at 37 °C and inoculated at 5% into LB medium. The culture was grown at 37 °C until the A600 was achieved. IPTG (final concentration 0.4 mM) was added to induce protein expression and cells were harvested 4h later and lysed by sonication. The inclusion bodies were pelleted by centrifugation for 20 min at 8000 × g, washed with washing buffer (20 mM Tris–HCl pH 8.0, containing 1% Triton-X100) followed by distilled water to remove contaminating salts and detergents. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ni-NTA agrose chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20 mM Tris–HCl pH 8.0, containing 1% Triton-X100 |
| Solubilization Buffer | 8M urea |
| Refolding Buffer | PBS (phosphate-buffered saline) |
| Pre-Refolding Purification | Ni-NTA agrose chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 0.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Recombinant EVM135 and A33R were purified under denaturing conditions by Ni-NTA metal affinity chromatography. The inclusion bodies were solubilized with 8M urea and loaded onto Ni-NTA agarose (Qiagen), according to the manufacturer\'s recommendations. After washing out the unbound proteins, the target protein was eluted by 0.5M imidazole in 8M Urea lysis buffer. The purified proteins were refolded by dialysis against PBS (phosphate-buffered saline). Protein concentrations were determined using a bicinchoninic acid assay (Pierce) with bovine serum albumin as a standard. The purity of each protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The same proteins were used for characterization, immunization and ELISA. |
| Refolding Assay | Immunoassay,ELISA |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |