Refolding Record:
| Protein | |
|---|---|
| Protein Name | B and T lymphocyte attenuator variant also known as BTLA protein |
| Abbreviated Name | BTLA |
| SCOP Family | TNF receptor-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q3HS85 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 241 |
| Molecular Weight | 27332.9 |
| Pi | 5.52 |
| Molecular Weight | 27332.9 |
| Disulphides | Unknown |
| Full Sequence |
MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEHSILAGDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGLNSRLARNVKEAPTEYASICVRS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Compaan DM, Gonzalez LC, Tom I, Loyet KM, Eaton D, Hymowitz SG. (2005) Biologycal Chemistry, 280, 39553-39561 |
| Project Aim | Structural Studies |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | n/a |
| Expression Protocol | DNA encoding human BTLA residues 26-137 (the initial methionine is residue 1) with the addition of a C-terminal His8 tag was expressed in Escherichia coli. Inclusion bodies from BTLA expressing E. coli were extracted under denaturing conditions, and the protein was purified on a Ni-NTA metal chelate column as described (19) |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | Ni-NTA agrose chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | n/a |
| Refolding Buffer | 0.1 M Tris (pH 8.6), 0.3 M NaCl, 20 mM glycine, 1 mM EDTA, 1 mM glutathione (oxidized), and 1 mM glutathione (reduced) |
| Pre-Refolding Purification | Ni-NTA agrose chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.6 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1/1 mM |
| Refolding Protocol | Fractions were pooled and diluted to 50 µg/ml with buffer containing 0.1 M Tris (pH 8.6), 0.3 M NaCl, 20 mM glycine, 1 mM EDTA, 1 mM glutathione (oxidized), and 1 mM glutathione (reduced). The refolding mixture was incubated overnight at 2-8 °C, and the pH adjusted to pH 3.0 with trifluoroacetic acid. The acidified refolding mixture was loaded onto an RP-HPLC Vydac C4 column (1.0 x 25 cm) equilibrated with 0.1% (w/v) trifluoroacetic acid in water and eluted with a linear gradient of acetonitrile (from 15 to 55%) in 0.1% trifluoroacetic acid at 3 ml/min for a total of 35 min. Protein fractions were pooled, and the acetonitrile was removed by evaporation assisted by a gentle stream of N2. The RP-HPLC pool was buffer exchanged using a HiTrap Desalting column (Amersham Biosciences) equilibrated with buffer containing 10 mM HEPES (pH 6.8), 0.15 M NaCl. BTLA activity was evaluated using SPR (Biacore). |
| Refolding Assay | Light scatter |
| Refolding Chaperones | None |
| Refolding Additives | Glycine |
| Additives Concentration | 20 mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |