Refolding Record:
| Protein | |
|---|---|
| Protein Name | Granzyme B |
| Abbreviated Name | GrB |
| SCOP Family | Eukaryotic Proteases |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P10144 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 243 |
| Molecular Weight | 27378.5 |
| Pi | 9.49496 |
| Molecular Weight | 27378.5 |
| Disulphides | 3 |
| Full Sequence |
MGSIEPD IIGGHEAKPH SRPYMAYLMI WDQKSLKRCG
GFLIQDDFVL TAAHCWGSSI NVTLGAHNIK EQEPTQQFIP VKRPIPHPAY NPKNFSNDIM LLQLERKAKR TRAVQPLRLP SNKAQVKPGQ TCSVAGWGQT APLGKHSHTL QEVKMTVQED RKCESDLRHY YDSTIELCVG DPEIKKTSFK GDSGGPLVCN KVAQGIVSYG RNNGMPPRAC TKVSSFVHWI KKTMKRY LNSHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lorentsen RH, Fynbo CH, Thogersen HC, Etzerodt M, Holtet TL (2005) Protein Expression and Purification, 39, 18-26 |
| Project Aim | Functional Studies |
| Fusion | C-terminal hexahis + pro seq |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 4 hrs |
| Expression Vector | pT7-rGrB-H6 |
| Expression Protocol | 1L of 2TY medium containing 100mg/ml ampicillin was incoulated wtih 30ml overnight culture and grown at 37degC with shaking until OD600 reached 0.7-0.8. Expression was induced by infection with bacteriophage lambda-CE6 at a multiplicity of approximately 5, and incubatgeion was continued for a furhter 4h before cells were harvested by centrifugation. |
| Method of Induction | Bacteriophage lambda |
| Cell Density at Induction | OD 600 = 0.7-0.8 |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | 0.5% (w/v) Triton X-100, 1mM EDTA |
| Solubilization Buffer | 6M GdnHCl, 50mM tris-HCl, pH8.0, 100mM DTT |
| Refolding Buffer | 50mM Tris-HCl, pH8.0, 0.5M NaCl, 2mM reduced glutathione, 0.2mM oxidised glutathione |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 10.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2mM/0.2mM,2mM/0.2mM |
| Refolding Protocol | The cell pellet from 3L culture was resuspended in 100ml lysis buffer )50mM TrisHCl pH 8.0, 25%(w/v) sucrose, 1mM EDTA before addition of lysozyme to a final concentration of 1mg/ml and incubation at room temperature for 15min. 200ml of detergent buffer containing 0.2M NaCl, 1%(w/v) deoxycholic acid, 1%(w/v) nonidet P-40, 20mM TrisHCl pH 7.5, 2mM EDTA was added and the preparation was gently homogenized before inclusion bodies were collected by centrifugation. The inclusionbodies were then washed at least 3 times in wash buffer before being dissolved in solubilization buffer. The protein preparation was then gel filtrated into a loading buffer containing 8M urea, 0.5M NaCl, 50mM TrisHCl, pH 8.0, 5mM 2-mercaptoethanol, before it was batch-mixed with Ni-NTA agarose for 5min and packed into a column. The protein was was purified by washing with 1 column volume of loading buffer followed by 1 column volume of 6M GdnHCl, 50mM TrisHCl, 5mM 2-mercaptoethanol and then loading buffer again until a stable baseline was reached. The column was then washed with a buffer containing 8M urea, 0.5M NaCl, 50mM TrisHCl, pH 8.0, 3mM reduced glutathione. The protein was then refolded in a cyclic fashion over a 24h period. Each cycle started with 45min of renaturation in refolding buffer followed by a step of denaturing and reducing conditions for 6 min, followed by a linear gradient of 8min from denaturation to renaturation and the start of the next cycle. The buffer applied in the denaturing phases was a mixed from the refolding buffer and a denaturation buffer containing 6M urea, 0.5M NaCl, 50mM TrisHCl, pH 8.0, 3mM glutathione. The percentage of denaturation buffer was decreased in steps from 100% in cycle 1 to 35% in cycle 23 before the procedure completed with 45min renaturation. The refolding was performed at 2ml/min flow rate and at 10degC. After completion of refolding, the column was washed with 2 column volumes of 0.5M NaCl, 50mM TrisHCl pH 8.0 before the refolded protein was eluted in the same buffer with 10mM EDTA. The eluted sample was then diluted with one volume of 50mM TrisHCl pH 7.0 and the pH adjusted to pH 7.0 with HCl. The protein was then loaded onto a SP Sepharose column pre-equilibrated in buffer containing 250mM NaCl, 50mM TrisHCl, pH 7.0 and fractionated by elution with a linear gradient from 250mM to 1M NaCl in 50mM TrisHCl pH 7.0 at room temperature. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | Not stated |
| Purity | 95% |
| Notes | Pro region removed by autocatalysis |