Refolding Record:
| Protein | |
|---|---|
| Protein Name | Pseudomonas sp. MIS38 Lipase |
| Abbreviated Name | PML |
| SCOP Family | Bacterial lipase |
| Structure Notes | |
| Organism | Pseudomonas sp. MIS38 |
| UniProt Accession | Q9RBY1 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 484 |
| Molecular Weight | 51415.2 |
| Pi | 4.89 |
| Molecular Weight | 51415.2 |
| Disulphides | 0 |
| Full Sequence |
MGVYDYKNFGTADSKALFSDAMAITLYSYHNLDNGFAAGYQHNGFGLGLPATLVTALLGGTDSQGVIPGIPWNPDSEKLALDAVKKAGWTPITASQLGYDGKTDARGTFFGEKAGYTTAQVEILGKYDAQGHLTEIGIAFRGTSGPRENLILDSIGDVINDLLAAFGPKDYAKNYVGEAFGNLLNDVVAFAKANGLSGKDVLVSGHSLGGLAVNSMADLSGGKWGGFFADSNYIAYASPTQSSTDKVLNVGYENDPVFRALDGSTFTGASVGVHDAPKESATDNIVSFNDHYASTAWNLLPFSILNIPTWISHLPTAYGDGMNRIIESKFYDLTSKDSTIIVANLSDPARANTWVQDLNRNAETHKGSTFIIGSDSNDLIQTFRDNGAFGQDRVVGFTLNDKLVFLGVQGVLPNDDFRAHASMVGQDTVLKFGGDSVTLVGVALNSLSADGIVIALAAALEIKRASPELAPEDPEPVEHHHHHH
|
| Notes | PML7 mutant lacks residues 405-543 and 381-396 |
| Expression | |
|---|---|
| Report | Kwon, H., Haruki, M., Morikawa, M., Omori, K., Kanaya, S. (2002) J Biosci Bioeng, 93, 157-164 |
| Project Aim | Functional 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 | HMS174(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3 |
| Expression Vector | pET-25b(+) |
| Expression Protocol | Cells were grown to an absorbance of 0.6 at 660nm before induction with 1 mM ITPG. Cells were then h |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 660 = 0.6 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 2 M urea, 2% Triton X-100 |
| Solubilization Buffer | 50 mM TrisHCl, pH 8.0 containing 1 mM EDTA, 5 % glycerol, 10 m M DTT and 8 M urea |
| Refolding Buffer | 50 mM TrisHCl, pH 7.5 containing 5 mM CaCl2 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a,n/a,n/a |
| Refolding Protocol | Precipitate was washed before being disolved in solubilization buffer. The resulting solution was then purified by application to a column of Hi-TrapQ that was equilibrated in the same buffer. PML was then eluted at a NaCL concentration of 0.15M in a single peak. Fractions were then dialyzed against refolding buffer. before application to a column of Superdex 200 previously equilibrated with 25 mM TrisHCl pH 7.5, containing 5 mM aCL2 and 0.15 NaCl. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |