Refolding Record:
| Protein | |
|---|---|
| Protein Name | NifU-like protein 1, chloroplast Also known as OsNifu1 |
| Abbreviated Name | OsNiful1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Oryza sativa |
| UniProt Accession | Q84LK7 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | OsNifU1A domain II |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 173 |
| Molecular Weight | 18354.6 |
| Pi | 8.98 |
| Molecular Weight | 18354.6 |
| Disulphides | Unknown |
| Full Sequence |
RPPLVVGAIAGLDPVTAVQLPLTAGNVESVLDQVRPYLTADGGDVALHEIAGNVVRLKLQGACGSCPSSLITIKRGIERRLMEKIPDVAAVEPVTDKETGLELNEENVEKVLNEIRPYLAGTGGGGLQFLMIKGPIVKVRLTGPAAVVRTVRIAVSKKLREKIPSIQIVQLLS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Katoh S, Murata K, Kubota Y, Kumeta H, Ogura K, Inagaki F, Asayama M, Katoh E. (2005) Protein Expression and Purification, 43, 149-56 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BLR(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pET-32a |
| Expression Protocol | For expression of recombinant OsNifU1A domain II fusion proteins, E. coli BL21 (DE3) competent cells (Novagen) were transformed with one of the expression vectors. Cells were grown at 37 °C in 500 mL of Luria–Bertani medium containing 50 μg/mL ampicillin (LBamp) to an absorbance of approximately 0.6 at 600 nm. Expression was induced by addition of isopropyl-1-thio-β-galctopyranoside (IPTG) to a final concentration of 1 mM. Cells were grown for an additional 3 h and then centrifuged at 5000g for 15 min. We found that these growth and induction conditions were optimal for protein purification. Cell pellets were stored at −80 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 20 mM Tris–HCl, pH 8.5, 6 M GuHCl, |
| Refolding Buffer | 20 mM Tris–HCl, pH 8.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 4 |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Insoluble cell lysate (inclusion bodies), isolated by centrifugation, was suspended in 20 mL of the buffer used for sonication and centrifuged to remove any remaining soluble material. The precipitate was dissolved in 20 mL of 20 mM Tris–HCl, pH 8.5, 6 M GuHCl, and mixed by inverting the solution for 4 h at room temperature. The solution was centrifuged at 27,000g for 30 min at 4 °C and the supernatant was diluted twofold with 20 mM Tris–HCl, pH 8.5. The same centrifugation conditions were used hereafter. The diluted supernatant was centrifuged after leaving it undisturbed for 4 h at room temperature. Supernatant was then dialyzed against 2 L of 20 mM Tris–HCl, pH 8.5, 1 M GuHCl for 16 h at 4 °C. For the Trx–(His)6 construct, dialysis tubing with a molecular weight cutoff of 14 kDa was used. For the (His)6 construct, dialysis tubing with a molecular weight cutoff of 3.5 kDa was used. The retentates for both fusion constructs were again centrifuged, but only the Trx–(His)6/OsNifU1A domain II fusion protein was purified further, because much of the (His)6 fusion protein had precipitated when GuHCl was removed. Purification involved Ni-chelation chromatography and gel filtration as described in the previous section. The purified Trx–(His)6 fusion protein was concentrated to 10 mL by centrifugation over a Centriplus YM-10 membrane (Millipore). To remove the region upstream of OsNifU1A domain II, 22 mg of the Trx–(His)6 fusion protein was digested with 125 U of Factor Xa (Novagene) for 16 h at 37 °C. The digestion mixture was eluted over Hi-Load 26/60 Superdex 75 pg. Pooled fractions, containing OsNifU1A domain II, were dialyzed against 2 L of 20 mM sodium phosphate (NaPi), pH 6.0, 100 mM NaCl, and 2.5 mM of 2-ME. After dialysis, all OsNifU1A domain II preparations were concentrated over a Centriplus YM-3 membrane. Additionally, 15N-labeled protein was concentrated further over a Centricon YM-3 membrane. |
| Refolding Assay | NMR analysis |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 36% |
| Purity | n/a |
| Notes | n/a |