Refolding Record:
| Protein | |
|---|---|
| Protein Name | Granzyme K |
| Abbreviated Name | GzmK |
| SCOP Family | Eukaryotic Proteases |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P49863 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 240 |
| Molecular Weight | 26109.1 |
| Pi | 9.49592 |
| Molecular Weight | 26109.1 |
| Disulphides | 4 |
| Full Sequence |
MEIIGG KEVSPHSRPF MASIQYGGHH VCGGVLIDPQ WVLTAAHCQY RFTKGQSPTV VLGAHSLSKN EASKQTLEIK
KFIPFSRVTS DPQSNDIMLV KLQTAAKLNK HVKMLHIRSK TSLRSGTKCK VTGWGATDPD SLRPSDTLRE VTVTVLSRKL CNSQSYYNGD PFITKDMVCA GDAKGQKDSC KGDSGGPLIC KGVFHAIVSG GHECGVATKP GIYTLLTKKY QTWIKSNLVP PHTN
|
| Notes | first two residues ME constitute pro-region of Granzyme K. Two other constructs also created, starting with MVIGG.... and MGEIIGG.... to investigate efficiency of cathepsin C cleavage versus processing within E.coli cells (see paper for more details) |
| Expression | |
|---|---|
| Report | Wilharm E, Parry MA, Friebel R, Tschesche H, Matschiner G, Sommerhoff CP, Jenne DE (1999) J Biol Chem, 274, 27331-27337 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | B834(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3-4h |
| Expression Vector | pET24c |
| Expression Protocol | Cells were grown in LB broth at 37degC until OD 600 reached 0.5-0.8. Expression was induced with 1mM IPTG and cells were grown a fruther 3-4h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5-0.8 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris-HCl, 60 mM EDTA, 1.5 M NaCl, 6% Triton X-100, pH 7.2 |
| Solubilization Buffer | 6 M guanidinium chloride, 100 mM Tris-HCl, 20 mM EDTA, 15 mM GSH, 150 mM GSSG, pH 8.0 |
| Refolding Buffer | 50 mM Tris-HCl, 0.5 M L-arginine, 20 mM CaCl2, 1 mM EDTA, 0.1 M NaCl, 0.5 mM L-cysteine |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.01-0.015mg/ml |
| Refolding Time | 48h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 0.5mM |
| Refolding Protocol | Bacteria were lysed in 50 mM Tris-HCl, 2 mM MgCl2 containing 10 µg/ml DNase I, and 0.25 mg/ml lysozyme, pH 7.2, by French press or sonification. IB were harvested by centrifugation and washed twice in 50 mM Tris-HCl, 60 mM EDTA, 1.5 M NaCl, 6% Triton X-100, pH 7.2, followed by two washing steps in 50 mM Tris-HCl, 60 mM EDTA, pH 7.2. Purified IB were solubilized in 6 M guanidinium chloride, 100 mM Tris-HCl, 20 mM EDTA, 15 mM GSH, 150 mM GSSG, pH 8.0, overnight at room temperature (RT) in an end-over-end rotator followed by dialysis against 6 M guanidinium chloride, pH 5.0, at 4 °C. Refolding was initiated by diluting solubilized proteins (usually 10-15 mg/ml) in 100 volumes of 50 mM Tris-HCl, 0.5 M L-arginine, 20 mM CaCl2, 1 mM EDTA, 0.1 M NaCl, 0.5 mM L-cysteine, pH 8.5, at RT, followed by an additional incubation period of 2 days at RT. The refolding solution was dialyzed against 100 volumes of PBS, pH 7.0, at 4 °C until conductivity of the dialyzed solution was equal to the dilution buffer. After centrifugation at 30,000g refolded proteins were filtrated and loaded onto a Mono S-Sepharose column (Amersham Pharmacia Biotech) in PBS and further purified by fast protein liquid chromatography using 20 column volumes of a linear salt gradient from 0.15 to 2 M NaCl in the same buffer at RT. GzmK concentrations were determined with the Bradford (Coomassie dye binding) assay. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.5M |
| Refolding Yield | |
| Purity | See gel. |
| Notes | |