Refolding Record:
| Protein | |
|---|---|
| Protein Name | Riboflavin synthase 2 beta chain |
| Abbreviated Name | RIbH2 |
| SCOP Family | Lumazine synthase |
| Structure Notes | |
| Organism | Brucella abortus |
| UniProt Accession | P61711 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 158 |
| Molecular Weight | 17355.9 |
| Pi | 6.58 |
| Molecular Weight | 17355.9 |
| Disulphides | Unknown |
| Full Sequence |
MNQSCPNKTSFKIAFIQARWHADIVDEARKSFVAELAAKTGGSVEVEIFDVPGAYEIPLHAKTLARTGRYAAIVGAAFVIDGGIYRHDFVATAVINGMMQVQLETEVPVLSVVLTPHHFHESKEHHDFFHAHFKVKGVEAAHAALQIVSERSRIAALV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Klinke S, Zylberman V, Vega DR, Guimarães BG, Braden BC, Goldbaum FA. (2005) J Mol Biol, 353, 124-127 |
| Project Aim | Crystallography |
| 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 | pET11b |
| Expression Protocol | The RibH2-encoding gene was cloned into the expression plasmid pET11b (Novagen, Madison, WI) as described.21 This construction was used to transform Escherichia coli BL21(DE3) competent cells (Stratagene, La Jolla, CA). Recombinant colonies were grown at 37 °C to an A600 of 1.0 in LB medium containing 100 μg/ml of ampicillin with agitation (300 rpm). A portion (5 ml) of this preparation was diluted to 500 ml and grown again until A600=1.0. At this point, the culture was induced by the addition of 1 mM IPTG and incubated for 4 h at 37 °C with agitation (300 rpm), leading to a high yield of RibH2 as inclusion bodies. The bacteria were centrifuged at 15,000g for 20 min at 4 °C and stored at −20 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 1.0 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50 mM Tris (pH 8.0), 5 mM EDTA |
| Solubilization Buffer | 50 mM Tris (pH 8.0), 5 mM EDTA, 8 M urea |
| Refolding Buffer | PBS containing 1 mM DTT |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 0.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 1 mM |
| Refolding Protocol | he bacterial pellet was suspended and sonicated in 50 mM Tris (pH 8.0), 5 mM EDTA, 1% (v/v) Triton X-100. Inclusion bodies were washed with solution lacking Triton X-100 and then solubilized at room temperature in 50 mM Tris (pH 8.0), 5 mM EDTA, 8 M urea, overnight with agitation. The material was dialyzed extensively against PBS containing 1 mM DTT with several changes of buffer, in order to achieve protein refolding. This preparation was purified by anion-exchange chromatography in an FPLC apparatus using a Mono-Q column (Amersham Biosciences, Uppsala, Sweden) equilibrated in buffer A (50 mM Tris (pH 8.5), 1 mM DTT). The fractions were eluted using a linear gradient of 0–1 M sodium chloride. The protein was then amidinated with 25 mM iodoacetamide at room temperature for 1 h in the dark in order to block its single free cysteine residue (Cys7). The protein was further purified by size-exclusion chromatography in a Superdex 200 column with isocratic elution in PBS. RibH2 was finally dialyzed in crystallization buffer (10 mM sodium/potassium phosphate (pH 5.5), 25 mM NaCl), concentrated by centrifugation to 10 mg/ml with Centricon YM-10 concentrators (Millipore, Billerica, MA) and stored at −20 °C. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |