Refolding Record:
| Protein | |
|---|---|
| Protein Name | Heparinase I also known as Heparin lyase I |
| Abbreviated Name | n/a |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Flavobacterium heparinum |
| UniProt Accession | Q05819 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 364 |
| Molecular Weight | 41337.6 |
| Pi | 9.31 |
| Molecular Weight | 41337.6 |
| Disulphides | Unknown |
| Full Sequence |
QQKKSGNIPYRVNVQADSAKQKAIIDNKWVAVGINKPYALQYDDKLRFNGKPSYRFELKAEDNSLEGYAAGETKGRTELSYSYATTNDFKKFPPSVYQNAQKLKTVYHYGKGICEQGSSRSYTFSVYIPSSFPDNATTIFAQWHGAPSRTLVATPEGEIKTLSIEEFLALYDRMIFKKNIAHDKVEKKDKDGKITYVAGKPNGWKVEQGGYPTLAFGFSKGYFYIKANSDRQWLTDKADRNNANPENSEVMKPYSSEYKTSTIAYKMPFAQFPKDCWITFDVAIDWTKYGKEANTILKPGKLDVMMTYTKNKKPQKAHIVNQQEILIGRNDDDGYYFKFGIYRVGNSTVPVTYNLSGYSETAR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Ernst S, Venkataraman G, Winkler S, Godavarti R, Langer R, Cooney CL, Sasisekharan R. (1996) Biochem J., 315, 589-597 |
| Project Aim | Expression and charactrization |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 2 h |
| Expression Vector | pET-3a, pET-12a, pET-15b |
| Expression Protocol | For each of the four expression constructs, 1 ng of plasmid was transformed in BL21(DE3) (Novagen), and a single colony was grown overnight and diluted to an absorbance of 0.06 unit in 50–400 ml of LB and 250 µg\\ml ampicillin or 30 µg\\ml kanamycin. (For -L28a, equally good expression was found when the colony was grown for 4–5 h to an absorbance of 2.5 units, concentrated 2-fold in medium and frozen with 15 % glycerol at k70 mC in aliquots of 1.3 ml, which could then be used directly as inoculum.) The culture was grown to an absorbance of 0.5 unit, induced with 1 mM isopropyl β--thiogalactopyranoside (IPTG ; Boehringer Mannheim, Indianapolis, IN, U.S.A.) for 2 h, harvested by centrifugation (4 mC ; 3500 g ; 10 min), washed in cold PBS and resuspended in 0.05 vol. of 50 mM Tris, 2 mM EDTA, pH 8.4, or (for -L15b and -L28a) in 20 mM Tris, 500 mM NaCl, 5 mM midazole, pH 7.9 (the binding buffer for nickel-chelate chromatography). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris, 2 mM EDTA, pH 8.4 |
| Solubilization Buffer | 4 M guanidine hydrochloride (GdHCl), 20 mM Tris, 500 mM NaCl, 5 mM imidazole, 0.5 % 2-mercaptoethanol, pH 7.9 |
| Refolding Buffer | 20 mM Tris, 500 mM NaCl, 5 mM imidazole, pH 7.9 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.9 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The insoluble fraction of the cell lysate was resuspended in 4 M guanidine hydrochloride (GdHCl), 20 mM Tris, 500 mM NaCl, 5 mM imidazole, 0.5 % 2-mercaptoethanol, pH 7.9, kept for 20 min at 4 mC and centrifuged at 12 000 g for 20 min. The supernatant contained the solubilized inclusion bodies and was refolded by dilution in 20 vol. of binding buffer (20 mM Tris, 500 mM NaCl, 5 mM imidazole, pH 7.9) for 2 h. Precipitates were separated by centrifugation, and the supernatant was filtered on a 0.45 µm sterile filter (Millipore, Bedford, MA, U.S.A.) before further purification. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |