Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cysteine proteinase B |
| Abbreviated Name | CPB |
| SCOP Family | Papain-like Cysteine Proteinases |
| Structure Notes | |
| Organism | Leishmania mexicana |
| UniProt Accession | P36400 |
| 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 | 451 |
| Molecular Weight | 48688.1 |
| Pi | 6.12324 |
| Molecular Weight | 48688.1 |
| Disulphides | 3 |
| Full Sequence |
HHHHHHHMATSRAALCAVAVVCVVLAAACAPARAIHVGTPAAALFEEFKRTYGRAYETLAEEQQRLANFERNLELMREHQARNPHAQFGITKFFDLSEAEFAARYLNGAAYFAAAKRHAAQHYRKARADLSAVPDAVDWREKGAVTPVKDQGACGSCWAFSAVGNIEGQWYLAGHELVSLSEQQLVSCDDMNDGCDGGLMLQAFDWLLQNTNGHLHTEDSYPYVSGNGYVPECSNSSELVVGAQIDG
HVLIGSSEKAMAAWLAKNGPIAIALDASSFMSYKSGVLTACIGKQLNHGVLLVGYDMTGEVPYWVIKNSWGGDWGEQGYVRVVMGVNACLLSEYPVSAHVRESAAPGTSTSSETPAPRPVMVEQVICFDKNCTQGCRKTLIKANECHKNGGGGASMIKCSPQKVTMCTYSNEFCVGGGLCFETPDGKCAPYFLGSIMNTCHYT
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Sanderson SJ, Pollock KG, Hilley JD, Meldal M, Hilaire PS, Juliano MA, Juliano L, Mottram JC, Coombs GH. (2000) Biochem J., 347, 383-388 |
| 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 | M15pREP4 |
| Expression Temp | 37.0 |
| Expression Time | 4.5h |
| Expression Vector | pQE-30 |
| Expression Protocol | Not reported |
| 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 | Triton X-100, 2M urea |
| Solubilization Buffer | 8M urea, 0.1M Tris-HCl, 10mM DTT, pH 8.0 |
| Refolding Buffer | 0.1M Tris/HCl, 5mM EDTA, 5mM cysteine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.1mg/ml |
| Refolding Time | 16h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The solubilised protein was initally dialysed against 0.1M Tris/HCl, 5mM EDTA, 5mM cysteine pH 7 overnight at 4C. Subsequently the protein was dialysed at 4C for 2h against 20mM Tris-HCl, 5mM EDTA pH 7.0 The protein was then purified on ion exchange. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 30% |
| Purity | |
| Notes | |