Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cathepsin B |
| Abbreviated Name | Cathepsin B |
| SCOP Family | Papain-like Cysteine Proteinases |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P07858 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 344 |
| Molecular Weight | 37821.6 |
| Pi | 5.87992 |
| Molecular Weight | 37821.6 |
| Disulphides | 6 |
| Full Sequence |
MWQLWASLCCLLVLANARSRPSFHPLSDELVNYVNKRNTTWQAGHNFYNVDMSYLKRLCG
TFLGGPKPPQRVMFTEDLKLPASFDAREQWPQCPTIKEIRDQGSCGSCWAFGAVEAISDR
ICIHTNAHVSVEVSAEDLLTCCGSMCGDGCNGGYPAEAWNFWTRKGLVSGGLYESHVGCR
PYSIPPCEHHVNGSRPPCTGEGDTPKCSKICEPGYSPTYKQDKHYGYNSYSVSNSEKDIM
AEIYKNGPVEGAFSVYSDFLLYKSGVYQHVTGEMMGGHAIRILGWGVENGTPYWLVANSW
NTDWGDNGFFKILRGQDHCGIESEVVAGIPRTDQYWEKI
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kuhelj R, Dolinar M, Pungercar J, Turk V. (1995) Eur J Biochem., 229, 533-539 |
| Project Aim | Structure-Function |
| Fusion | None |
| 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 | 3h |
| Expression Vector | pET3a |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM Tris-HCl, 5mM EDTA, 2M urea pH 8.0 |
| Solubilization Buffer | 8M urea, 0.1M Tris-HCl pH 8.0 |
| Refolding Buffer | 0.1M sodium phosphate, 5mM cysteine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | <25 micrograms/ml |
| Refolding Time | 16h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The inclusion body pellet was washed once with 50mM Tris-HCl, 5mM EDTA, 0.1% Triton X-100 pH 8.0 and twice with 50mM Tris-HCl, 5mM EDTA, 2M urea pH 8.0. The protein was then subjected to sulphonation and unfolded with solublization buffer. The protein was refolded overnight at 4 degrees C by dialysis. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 3mg/L culture |
| Purity | |
| Notes | |