Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lipase |
| Abbreviated Name | lip4 |
| SCOP Family | Fungal lipases |
| Structure Notes | |
| Organism | Burkholderia cepacia KWI-56 |
| UniProt Accession | P22088 |
| 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 | 321 |
| Molecular Weight | 33174.1 |
| Pi | 5.56 |
| Molecular Weight | 33174.1 |
| Disulphides | Unknown |
| Full Sequence |
AAGYAATRYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQDVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAIQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALYGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVIRTHANRLKLAGV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Singh PK, Gupta MN. (2008) Biochemica et Biophysica Acta, 1, Epub ahead of p |
| Project Aim | Protein refolding |
| Fusion | C-terminal intein |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | E coli |
| Expression Strain | ER2566 |
| Expression Temp | 37.0 |
| Expression Time | 12 |
| Expression Vector | pTYB12 |
| Expression Protocol | The recombinant pTYB12-lip A (lipase gene from Burkholderia cepacia was cloned in pTYB12 vector as described previously [19]) was transformed into competent E. coli ER2566 cells and resulting recombinant E. coli cells were grown overnight at 37 °C on Luria–Bertani (LB) agar (1% tryptone, 0.5% yeast extract, 0.5% NaCl and 2% agar powder) containing 100 μg/ml ampicillin. Ten milliliters of LB media supplemented with 100 μg/ml ampicillin was inoculated with one of the resulting colonies and incubated overnight at 37 °C, under shaking conditions (200 rpm). One percent of the primary inoculum of the cul t ure was transferred to 50 0 ml of fresh LB medium supplemented with 100 μg/ml ampicillin. The cells were allowed to grow under shaking conditions (200 rpm) at 37 °C. Cultures were induced with 0.3 mM isopropyl-β-D-thiogalacto-pyranoside (IPTG) in the logarithmic phase (absorbance at 600 nm of 0.6–0.8) and then incubated at 37 °C for 12 h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6-0.8 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Chitin Column Chromatography |
| Wash Buffer | 20 mM HEPES, pH 8.0, containing 500 mM NaCl, 10 mM EDTA and 1% Triton X- 100 |
| Solubilization Buffer | 20 mM HEPES, pH 8.0, containing 500 mM NaCl, 10 mM dithiothreitol (DTT) and 8 M urea or 5% cationic surfactant, cetyltrimethylammonium bromide (CTAB) |
| Refolding Buffer | 50 mM DTT (in 20 mM HEPES, pH 8.0, containing 0.5 M NaCl) |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 20.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The inclusion bodies were finally solubilized in 20 mM HEPES, pH 8.0, containing 500 mM NaCl, 10 mM dithiothreitol (DTT) and 8 M urea or 5% cationic surfactant, cetyltrimethylammonium bromide (CTAB) in 0.1 M glycine/HCl, pH 10.0. CTAB was removed by ion exchange chromatography using Dowex 50WX4 cation exchange resin [20]. The CTAB free supernatant was dialyzed against 20 mM HEPES, pH 8.0, containing 500 mM NaCl, 10 mM DTT and 8 M urea. For purification on chitin beads [21] the solubilized inclusion bodies were first diluted to 4 M urea with 20 mM HEPES, pH 8.0, containing 500 mM NaCl. The solution was then passed through a column packed with chitin beads (1 ml packed bed volume) at 4 °C and the column was immediately washed with 5 bed volumes of 20 mM HEPES, pH 8.0, containing 500 mM NaCl. After extensive washing of the column with buffer, a solution of 50 mM DTT (in 20 mM HEPES, pH 8.0, containing 0.5 M NaCl) was passed through the column. The flow was stopped and the column was transferred to 25 °C. This step involving treatment with DTT cleaves the intein tag and frees the protein (lipase) from the bound state. After incubation for 16 h, the protein was then eluted in 20 mM HEPES, pH 8.0, containing 0.5 M NaCl. The eluted proteins were dialyzed against 50 mM Tris–HCl buffer, pH 7.0 to remove the DTT. Enzyme assay was carried out to estimate the lipase activity. |
| Refolding Assay | Fluorescence,Folding Kinetics,Circular Dichroism (uv-CD) |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 82% |
| Purity | n/a |
| Notes | n/a |