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 | 646 |
| Molecular Weight | 67790.0 |
| Pi | 4.7 |
| Molecular Weight | 67790.0 |
| Disulphides | 0 |
| Full Sequence |
MGVYDYKNFG TADSKALFSD AMAITLYSYH NLDNGFAAGY QHNGFGLGLP ATLVTALLGG TDSQGVIPGI PWNPDSEKLA LDAVKKAGWT PITASQLGYD GKTDARGTFF GEKAGYTTAQ VEILGKYDAQ GHLTEIGIAF RGTSGPRENL ILDSIGDVIN DLLAAFGPKD YAKNYVGEAF GNLLNDVVAF AKANGLSGKD VLVSGHSLGG LAVNSMADLS GGKWGGFFAD SNYIAYASPT QSSTDKVLNV GYENDPVFRA LDGSTFTGAS VGVHDAPKES ATDNIVSFND HYASTAWNLL PFSILNIPTW ISHLPTAYGD GMNRIIESKF YDLTSKDSTI IVANLSDPAR ANTWVQDLNR NAETHKGSTF IIGSDSNDLI QGGSGNDYLE GRAGNDTFRD GGGYNVILGG AGNNTLDLQK SVNTFDFAND GAGNLYVRDA NGGISITRDI GSIVTKEPGF LWGLFKDDVT HSVTASGLKV GSNVTQYDAS VKGTNGADTL KAHAGGDWLF GLDGNDHLIG GVGNDVFVGG AGNDLMESGG GADTFLFNGA FGQDRVVGFTL NDKLVFLGV QGVLPNDDFR AHASMVGQDT VLKFGGDSVT LVGVALNSLS ADGIVIA LAAALEIKRASPELAPEDPEPVEHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Amada. K., Haruki, M., Imanaka, T., Morikawa, M., Kanaya, S. (2000) Acta Biochemica et Biophysica Sinica, 1478, 201-210 |
| 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 |