Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cystic fibrosis transmembrane conductance regulator |
| Abbreviated Name | CFTR |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P13569 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | CFTR NBD |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 563 |
| Molecular Weight | 63428.6 |
| Pi | 6.09 |
| Molecular Weight | 63428.6 |
| Disulphides | Unknown |
| Full Sequence |
TEVVMENVTAFWEEGFGELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAGKTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHRFSLKDDIWPSGGQMTVKDLTAKYTEGGNAILENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEGEIQIDGVSWDSITLQQWRKAFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Gross CH, Abdul-Manan N, Fulghum J, Lippke J, Liu X, Prabhakar P, Brennan D, Willis MS, Faerman C, Connelly P, Raybuck S, Moore J. (2006) Biologycal Chemistry, 281, 4058-4068 |
| Project Aim | Undefined |
| 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)pLysS |
| Expression Temp | 25.0 |
| Expression Time | 00 |
| Expression Vector | pET28b |
| Expression Protocol | Expression, Purification, and Refolding of the CFTR NBD Proteins—Mouse and human CFTR NBD clones were expressed in E. coli using BL21 DE3 pLysS cells induced with 0.5 mM isopropyl-1-thio-B-D-galactopyranoside at 25 °C. Soluble mouse NBD1 was initially purified by nickel ion affinity chromatography, followed by a Sepharose S200 (16/60 mm column) sizing chromatography step; the Smt3 tag then was removed using the Ulp1 protease (19). Subsequently, a second nickel affinity step was employed to separate the tag from the cleaved mNBD1 protein. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 25 mM Tris-HCl, pH 7.5, 8 M urea, and 10 mM 2-mercaptoethanol |
| Solubilization Buffer | 25 mM Tris-HCl, pH 7.5, 6 M guanidine hydrochloride, and 10 mM 2-mercaptoethanol |
| Refolding Buffer | 25 mM Tris-HCl, pH 7.0, 1 mM EDTA, 10 mM -cyclodextrin, 0.1 mM GSH/0.01 mM GSSG, and 0.5 M NDSB-256 |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 0.1/0.01 mM |
| Refolding Protocol | The human NBD proteins were found in the insoluble fraction. The cell pellets (30 g) were resuspended in 600 ml of buffer 1 (25 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.1% Tween-20, and 10 mM 2-mercaptoethanol) followed by the addition of protease inhibitors (E-64, pepstatin, and leupeptin at 2 µg/ml, DFP at 50 µM). The cell pellets were dounce-homogenized and incubated for 1 h at 4°C. Samples were spun at 54,000 x g for 1 h at 4 °C. Supernatants were removed, and the pellets were resuspended in 600 ml of buffer 2 (25 mM Tris-HCl, pH 7.5, 6 M guanidine hydrochloride, and 10 mM 2-mercaptoethanol) overnight on a magnetic stirrer at 4 °C. Samples were spun at 54,000 x g for 1 h at 4 °C. To the supernatants, 30 ml of Nickel Hi-Selects resin (Sigma) were added and incubated overnight at 4 °C. Samples were centrifuged to pellet resin and resuspended in buffer 2 and poured into a column. The packed column was washed with buffer 3 (25 mM Tris-HCl, pH 7.5, 8 M urea, and 10 mM 2-mercaptoethanol) containing sequentially increasing amounts of imidazole. Fractions were analyzed using SDS-PAGE and Coomassie Blue staining. Fractions containing protein were concentrated by Centricon (Amicon) and loaded onto Superdex S-200 16/60 mm column. Proteins were eluted in buffer 4 (25 mM Tris-HCl, pH 7.5, 8 M urea, 150 mM NaCl, and 10 mM 2-mercaptoethanol). Protein containing fractions were concentrated to 1 mg/ml. Protein concentrations were determined from A280 using calculated extinction coefficients (20). The initial \"in house\" NDSB-256 (dimethyl benzyl ammonium propane sulfonate) (Anatrace Inc.) refolding buffer was (25 mM Tris-HCl, pH 7.0, 1 mM EDTA, 10 mM -cyclodextrin, 0.1 mM GSH/0.01 mM GSSG, and 0.5 M NDSB-256). The refolding screen for the denatured proteins has been described (21). Larger batches (10 mg) of purified proteins were refolded (hNBD1 100 µg/ml, hNBD2 50 µg/ml) in either condition 10 (21), condition 10 plus 0.5 M NDSB-256 or an NDSB-256 buffer from above. After 24 h incubation at 4 °C, samples were concentrated, filtered and sized on a Superdex S-200 10/30 mm column. Sizing fractions that yielded monomeric/dimeric NBD proteins were pooled. Protein concentrations of the refolded samples were determined by quantitative Westerns using specific monoclonal antibodies (hNBD1 clone L12B4) and (hNBD2 clone M3A7) (Chemicon Inc.) and known quantities of denatured hNBD proteins as a standard curve. Western detection was accomplished using infrared fluorescence-labeled secondary antibody and the Odyssey system (LI-COR Bioscience) (22). |
| Refolding Assay | Adenylate Kinase and ATPase assay |
| Refolding Chaperones | None |
| Refolding Additives | α-cyclodextrin |
| Additives Concentration | 10 mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |