Refolding Record:
| Protein | |
|---|---|
| Protein Name | Non-structural glycoprotein 4 |
| Abbreviated Name | NSP4 |
| SCOP Family | NSP4 oligomerization domain |
| Structure Notes | |
| Organism | Simian rotavirus |
| UniProt Accession | P04512 |
| 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 | 437 |
| Molecular Weight | 49216.4 |
| Pi | 6.37 |
| Molecular Weight | 49216.4 |
| Disulphides | Unknown |
| Full Sequence |
MEKLTDLNYTLSVITLMNNTLHTILEDPGMAYFPYIASVLTGLFALNKASIPTMKIALKTSKCSYKVVKYCIVTIFNTLLKLAGYKEQITTKDEIEKQMDRVVKEMRRQLEMIDKLTTREIEQVELLKRIYDKLTVQTTGEIDMTKEINQKNVRTLEEWESGKNPYEPREVTAAMADVCMDPEPIVRIVGRNGLCVDVRDGRFHNGNAIQLWPCKSNTDANQLWTLKRDNTIRSNGKCLTTYGYSPGVYVMIYDCNTAATDATRWQIWDNGTIINPRSSLVLAATSGNSGTTLTVQTNIYAVSQGWLPTNNTQPFVTTIVGLYGLCLQANSGQVWIEDCSSEKAEQQWALYADGSIRPQQNRDNCLTSDSNIRETVVKILSCGPASSGQRWMFKNDGTILNLYSGLVLDVRASDPSLKQIILYPLHGDPNQIWLPLF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Choi NW, Estes MK, Langridge WH (2006) J Biotechnology, 121, 272-283 |
| Project Aim | Vaccine studies |
| 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 | 3-4 h |
| Expression Vector | pLysS |
| Expression Protocol | Purification of the fusion proteins produced in E. coli was performed under denaturation conditions in 8 M urea (Nagahara et al., 1998). Briefly, a bacterial colony harboring the recombinant plasmid was inoculated into 25 ml of LB medium containing ampicillin, 100 mg/l and incubated overnight at 37 °C. The culture was transferred to 250 ml of LB medium and incubation continued until the culture OD600 reached approximately 0.5. To maximize the recombinant protein expression levels, isopropyl β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to stimulate recombinant protein synthesis. The bacterial cultures were incubated for an additional 3–4 h prior to harvest by centrifugation (4000 ×g, 10 min, 4 °C). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 8 M urea, 100 mM NaCl, 20 mM HEPES, pH 8.0, imidazole 20 mM |
| Solubilization Buffer | 8 M urea, 100 mM NaCl, 20 mM HEPES, pH 8.0, imidazole 250 mM |
| Refolding Buffer | 40 mM galactose, 1 mM NaCl and 0.1 mM DTT |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 0.1 mM,0.1 mM |
| Refolding Protocol | The cells were lysed in 10 ml of buffer “Z” (8 M urea, 100 mM NaCl, 20 mM HEPES, pH 8.0) by sonication (60 Sonic Dismembrator, Fisher Scientific) on ice 3 times for 10 s with output power of 10 W. After centrifugation at 12,000 ×g, 10 min, 4 °C in SS34 rotor in Sorvall RC5 refrigerated centrifuged to remove cell debris, imidazole was added to the bacterial lysate supernatant to a final concentration of 10 mM and the sample loaded onto a 2 ml nickel column (Ni-NTA) (Invitrogen, Carlsbad, CA). The histidine affinity column was washed with 30 ml of Buffer Z plus imidazole (20 mM) to remove weakly bound proteins of E. coli origin. The His-tagged recombinant proteins were eluted with buffer Z plus 250 mM imidazole. The purified recombinant proteins were quantified by Bradford protein assay (Bio-Rad, Hercules, CA) and dialyzed in several different concentrations of galactose, salt (NaCl) and reducing agent (DTT) to remove the urea and imidazole. Optimal conditions for purification of biologically active forms of RTB and NSP490–RTB fusion proteins were determined by removal of urea by dialysis in the presence of 80 mM galactose, 1 mM NaCl and 0.1 mM DTT for the RTB and by dialysis in 40 mM galactose, 1 mM NaCl and 0.1 mM DTT for the NSP490–RTB fusion protein. After dialysis, the RTB and NSP490–RTB fusion proteins were analyzed by separation on 10% gels by SDS-polyacrylamide gel electrophoresis (PAGE) (Bio-Rad, Cat. 165-3302). The recombinant RTB and NSP490–RTB fusion proteins were detected on immunoblots by incubation with primary rabbit anti-His tag antibody (Santa Cruz Biotechnology, 1:3000 dilution) followed by washing and incubation with a secondary alkaline phosphatase-conjugated mouse anti-rabbit IgG (Sigma, 1:20,000 dilution). |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |