Refolding Record:
| Protein | |
|---|---|
| Protein Name | Procathepsin L |
| Abbreviated Name | Cathepsin L |
| SCOP Family | Papain-like Cysteine Proteinases |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P07711 |
| 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 | 338 |
| Molecular Weight | 37564.1 |
| Pi | 5.31542 |
| Molecular Weight | 37564.1 |
| Disulphides | 3 |
| Full Sequence |
MNPTLILAAFCLGIASATLTFDHSLEAQWTKWKAMHNRLYGMNEEGWRRAVWEKNMKMIE
LHNQEYREGKHSFTMAMNAFGDMTSEEFRQVMNGFQNRKPRKGKVFQEPLFYEAPRSVDW
REKGYVTPVKNQGQCGSCWAFSATGALEGQMFRKTGRLISLSEQNLVDCSGPQGNEGCNG
GLMDYAFQYVQDNGGLDSEESYPYEATEESCKYNPKYSVANDTGFVDIPKQEKALMKAVA
TVGPISVAIDAGHESFLFYKEGIYFEPDCSSEDMDHGVLVVGYGFESTESDNNKYWLVKN
SWGEEWGMGGYVKMAKDRRNHCGIASAASYPTV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Dolinar M, Maganja DB, Turk V.Dolinar M, Maganja DB, Turk V. (1995) Biol Chem Hoppe Seyler., 376, 385-388 |
| Project Aim | Undefined |
| 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 | 4h |
| Expression Vector | pET81F+ |
| 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, 2M urea, pH 8. |
| Solubilization Buffer | 7M urea, 100mM Tris-HCl, pH 8 |
| Refolding Buffer | 00mM Tris-HCl, 0.015% Triton X-100, 3mM cysteine, 0.1mM cystine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.6 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.06mg/ml |
| Refolding Time | |
| Redox Agent | Cysteine/Cystine |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The inclusion bodies were washed once with 50mM Tris-HCl, 0.1% Triton X-100, pH 8 and subsequently with 50mM Tris-HCl, 2M urea, pH 8. The pellet was solubilized and sulphonated in 7M guanidinium chloride, 0.3M Na2SO3 containing disodium 2-nitro-5-thiosulfobenzoate and precipitated in ice cold 1% acetic acid. The precipitate was dissolved in 7M urea, 100mM Tris-HCl, pH 8 and applied to gel filtration. The proteins were eluted in 6M urea, 3mM EDTA, 50mM Tris-HCl, pH 8. The protein was refolded by dialysis against 100mM Tris-HCl, 0.015% Triton X-100, 3mM cysteine, 0.1mM cystine, pH 8.6. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 4.4%- 0.15mg/L culture |
| Purity | pure |
| Notes | |