Refolding Record:
| Protein | |
|---|---|
| Protein Name | Rapamycin-binding protein |
| Abbreviated Name | FKBP |
| SCOP Family | FKBP immunophilin/proline isomerase |
| Structure Notes | |
| Organism | S. cerevisiae |
| UniProt Accession | P20081 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 114 |
| Molecular Weight | 12157.9 |
| Pi | 5.72 |
| Molecular Weight | 12157.9 |
| Disulphides | Unknown |
| Full Sequence |
MSEVIEGNVKIDRISPGDGATFPKTGDLVTIHYTGTLENGQKFDSSVDRGSPFQCNIGVGQVIKGWDVGIPKLSVGEKARLTIPGPYAYGPRGFPGLIPPNSTLVFDVELLKVN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Dames SA. (2008) Protein Expression and Purification, 1, 1 |
| Project Aim | Protein refolding |
| Fusion | N-terminal GST |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | Rosetta (DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pGEX-4T |
| Expression Protocol | The yeast FKBP-homolog FPR1 was expressed as N-terminal fusion to GST from E. coli Rosetta (DE3) cells (Novagen). Fifty milliliters of LB medium supplemented with 100 μg/ml ampicillin and 40 μg/ml chloramphenicol were inoculated with a single colony of transformed cells and grown overnight at 30 °C. The starter culture was diluted 1:40 in 1.6 L LB medium supplemented with 100 μg/ml ampicillin and 40 μg/ml chloramphenicol. The prepared culture was grown at 37 °C to an OD600 of ≈1.0 and induced with 0.5 mM IPTG. The cells continued growing for another 3 h at 37 °C. Cells expressing FPR1 were harvested by centrifugation at 4 °C (20 min, 5000 rpm, Sorvall SLA-3000 rotor). The cell pellet was resuspended in 50 ml lysis buffer (1× PBS = 10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 2.8 mM KCl, pH 7.2). The cell suspension was supplemented with 1 ml of lysozyme stock solution (10 mg/ml) and incubated for 10 min at room temperature. Cells were disrupted by sonication on ice using a Branson Digital Sonifier and a power level of 40%. During sonication for 5 min intervals of 3 s pulsing alternated with 7 s breaks. To verify that FPR1 is in the soluble fraction 100 μl of the sonicated cell suspension were centrifuged and the supernatant and the pellet checked by SDS–PAGE |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 1.0 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | RP-HPLC |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | n/a |
| Refolding Buffer | 20 mM Tris, 300 mM NaCl, 1 mM TCEP, pH 8 |
| Pre-Refolding Purification | RP-HPLC |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | After that the whole sonicated cell suspension was centrifuged for 30 min at 12,000 rpm (Sorvall SS34 rotor). The supernatant was loaded on a glutathione sepharose™ 4B column according to the manufacture’s manual (Amersham Biosciences). The GST-fusion protein was extracted with 50 mM Tris, 10 mM reduced glutathione, pH 8. The fractions containing GST-FPR1 were dialyzed to 1× PBS and digested with thrombin for 5 days at room temperature (20 U per 1 ml with 0.1 mM fusion protein). The cleaved GST was removed by RP HPLC, which was performed as described for hFRB but using a preparative C8 column. FPR1 eluted around 48–51%. Fractions were analyzed by SDS–PAGE and those containing nearly pure FPR1 were frozen in liquid nitrogen, lyophilized and stored at −20 °C. The lyophilized protein was refolded by dissolving it in 10 ml of 20 mM Tris, 300 mM NaCl, 1 mM TCEP, pH 8 to obtain about 20 μM solution and concentrated using an ultrafiltration spin column (Vivaspin 20, Vivascience, MWCO 5000). That the protein adapted its native structure after refolding was confirmed from a comparison of its 1D 1H NMR spectrum with the one of the protein purified under native conditions using a 2nd glutathione sepharose™ 4B column after cleavage with thrombin. To have the protein purified in both ways in the same buffer, the one collected in the flow-through of the glutathione sepharose™ 4B column after thrombin cleavage was concentrated to about 0.5 ml using an ultrafiltration spin column (Vivaspin 20, Vivascience, MWCO 5000) and washed 2× with 17 ml of 20 mM Tris, 300 mM NaCl, 1 mM TCEP, pH 8. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |