Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lipase from Rhizopus oryzae |
| Abbreviated Name | ROL |
| SCOP Family | Fungal lipases |
| Structure Notes | |
| Organism | Rhizopus oryzae |
| UniProt Accession | P61872 |
| 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 | 309 |
| Molecular Weight | 33826.5 |
| Pi | 8.76533 |
| Molecular Weight | 33826.5 |
| Disulphides | 3 |
| Full Sequence |
MKKTAIAIAVALAGFATVAQAAPKDNTWYTGAKLGWSQYH
SDGGKVV AATTAQIQEF TKYAGIAATA YCRSVVPGNK WDCVQCQKWV PDGKIITTFT SLLSDTNGYV LRSDKQKTIY
LVFRGTNSFR SAITDIVFNF SDYKPVKGAK VHAGFLSSYE QVVNDYFPVV QEQLTAHPTY KVIVTGHSLG GAQALLAGMD LYQREPRLSP KNLSIFTVGG PRVGNPTFAY YVESTGIPFQ RTVHKRDIVP HVPPQSFGFL HPGVESWIKS GTSNVQICTS EIETKDCSNS IVPFTSILDH LSYFDINEGS CL
|
| Notes | PreProROL and ProROL also expressed and refolded, to investigate role or leader sequences. PreProROL: MKKTAIAIAVALAGFATVAQAAPKDNTWYTGAKLGWSQYH MVSFISISQG VSLCLLVSSM MLGSSAVPVS GKSGSSNTAV SASDNAALPP 50 LISSRCAPPS NKGSKSDLQA EPYNMQKNTE WYESHGGNLT SIGKRDDNLV 100 GGMTLDLPSD APPISLSSST NSASDGGKVV AATTAQIQEF TKYAGIAATA 150 YCRSVVPGNK WDCVQCQKWV PDGKIITTFT SLLSDTNGYV LRSDKQKTIY 200 LVFRGTNSFR SAITDIVFNF SDYKPVKGAK VHAGFLSSYE QVVNDYFPVV 250 QEQLTAHPTY KVIVTGHSLG GAQALLAGMD LYQREPRLSP KNLSIFTVGG 300 PRVGNPTFAY YVESTGIPFQ RTVHKRDIVP HVPPQSFGFL HPGVESWIKS 350 GTSNVQICTS EIETKDCSNS IVPFTSILDH LSYFDINEGS CL Number of amino acids: 432 Molecular weight: 46372.5 Theoretical pI: 8.30 ProROL: MKKTAIAIAVALAGFATVAQAAPKDNTWYTGAKLGWSQYH AVPVS GKSGSSNTAV SASDNAALPP 50 LISSRCAPPS NKGSKSDLQA EPYNMQKNTE WYESHGGNLT SIGKRDDNLV 100 GGMTLDLPSD APPISLSSST NSASDGGKVV AATTAQIQEF TKYAGIAATA 150 YCRSVVPGNK WDCVQCQKWV PDGKIITTFT SLLSDTNGYV LRSDKQKTIY 200 LVFRGTNSFR SAITDIVFNF SDYKPVKGAK VHAGFLSSYE QVVNDYFPVV 250 QEQLTAHPTY KVIVTGHSLG GAQALLAGMD LYQREPRLSP KNLSIFTVGG 300 PRVGNPTFAY YVESTGIPFQ RTVHKRDIVP HVPPQSFGFL HPGVESWIKS 350 GTSNVQICTS EIETKDCSNS IVPFTSILDH LSYFDINEGS Number of amino acids: 405 Molecular weight: 43597.0 Theoretical pI: 8.37 |
| Expression | |
|---|---|
| Report | Beer HD, McCarthy JEG, Bornscheuer UT, Schmid RD (1998) Biochim Biophys Acta., 1399, 173-180 |
| Project Aim | Structure-Function |
| Fusion | N-terminal OmpA signal sequence |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL321 |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pCYTEXP1 |
| Expression Protocol | Cells were incubated in LB medium, when OD600 reached 0.5-1, the cultures were shifted from 30degC to 42degC and incubated a further 3h. Cells were harvested by centrifugation (15min, 3000g). |
| Method of Induction | Temperature Shift |
| Cell Density at Induction | OD 600 = 0.5-1 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 1: 100mM NaCl, 50mM TrisHCl, 1mM EDTA pH 8, 2: 100mM NaCl, 50mM TrisHCl, 10mM EDTA, 0.5% Triton X-100, pH 8 |
| Solubilization Buffer | 5M GdnHCl, 50mM TrisHCl pH 8.5, 30mM DTT |
| Refolding Buffer | 50mM TrisHCl pH 8.5, 2mM CaCl2, 10% glycerol, 2mM reduced glutathione, 1mM oxidized glutathion |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 36h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2mM/1mM,2mM/1mM,2mM/1mM |
| Refolding Protocol | The cells of two 250ml cultures were then resuspended in 16ml lysis buffer (50mM TrisHCl, 1mM EDTA, pH 8) and sonicated for 7 min. After centrifugation(20min, 17000g) the pellet was resuspended in 16ml Wash buffer 1 and incubated for 20min at room temperature in the presence of 30micrograms/ml of DNAse. After centrifugation, the pellet was washed with Wash buffer 2 and then again with Wash Buffer I. The pellet was then resuspended in 16ml Solubilization buffer and incubated at room temperature for 30min. The mixture was then centrifuged (30min, 4degC, 17000g) and the supernatant was then diluted in 1L Refolding buffer. After 36h at 10degC, the solution was dialyzed against 10mM CaCl2, centrifuged (20min, 4degC, 10000g) and filtered (45micron). The solution was then applied to an S-sepharose column and eluted with a linear gradient of 0-500mM NaCl. The fractions containing protein were pooled, dialysed against deionized water and freeze-dried. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 10% |
| Refolding Yield | |
| Purity | |
| Notes | See also Beer et al (1996) Biochem J v.319, 351-359 |