Refolding Record:
| Protein | |
|---|---|
| Protein Name | ProCathepsin K |
| Abbreviated Name | ProCathepsin K |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P43235 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 324 |
| Molecular Weight | 36565.3 |
| Pi | 8.72453 |
| Molecular Weight | 36565.3 |
| Disulphides | 3 |
| Full Sequence |
MRGSHHHHHH LYPEE ILDTHWELWK KTHRKQYNNK VDEISRRLIW EKNLKYISIHNLEASLGVHT YELAMNHLGD MTSEEVVQKM TGLKVPLSHS RSNDTLYIPE WEGRAPDSVD YRKKGYVTPV KNQGQCGSCW AFSSVGALEG QLKKKTGKLL NLSPQNLVDC VSENDGCGGGYMTNAFQYVQ KNRGIDSEDA YPYVGQEESC MYNPTGKAAK CRGYREIPEG NEKALKRAVA RVGPVSVAID ASLTSFQFYS KGVYYDESCN SDNLNHAVLA VGYGIQKGNK HWIIKNSWGENWGNKGYILM ARNKNNACGI ANLASFPKM
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hwang HS, Chung HS. (2002) Protein Expression and Purification, 25, 541-546 |
| Project Aim | Functional Studies |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JM109 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pQE-30 |
| Expression Protocol | The E. coli strain JM109 was transformed with the pQE-30-human procathepsin K and then grown to a density of OD600=0.6–0.8 in 100 ml LB medium supplemented with 50 microg/ml ampicillin. The expression of procathepsin K was induced by adding IPTG to a final concentration of 1 mM. After 4 h at 37degC, cell pellet was collected by centrifugation at 10,000g for 20 min, |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 50mM Tris-HCl, 500mM NaCl, 2M urea pH 8.0 |
| Solubilization Buffer | 50mM Tris-HCl, 500mM NaCl, 6M urea, 5mM imidazole pH 8.0 |
| Refolding Buffer | 50mM Tris-HCl, 500mM NaCl, 5mM EDTA, 10mM GSH, 1mM GSSG, 0.7M L-arginine, 1% CHAPS |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 60 micrograms/ml |
| Refolding Time | 12h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 10mM/1mM |
| Refolding Protocol | Inclusion bodies were stirred in solubilization buffer for 1h at 4C and clarified using centrifugation and filtration. Protein was then purified on Ni-NTA. Purified protein was refolded first by 50-fold dilution into refolding buffer and incubated overnight at 4C with stirring. Protein was subsequently concentrated and dialysed agains 25mM Tris-HCl, 500mM NaCl pH 8.0. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.7M |
| Refolding Yield | 3.5mg from 100ml culture |
| Purity | see gel attached |
| Notes | |