Refolding Record:
| Protein | |
|---|---|
| Protein Name | Carboxypeptidase Y |
| Abbreviated Name | CPY |
| SCOP Family | Serine carboxypeptidase-like |
| Structure Notes | |
| Organism | Yeast (Candida albicans) |
| UniProt Accession | P30574 |
| 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 | 551 |
| Molecular Weight | 61044.2 |
| Pi | 5.29118 |
| Molecular Weight | 61044.2 |
| Disulphides | 5 |
| Full Sequence |
MKLSKSTLIATLALTATSTNALVVQNPFSNIQQALKLDLSYDKLTSKLTDTFEQGKANII
STIAKVMNEPLDGLTPEIKNIWSEMLMKFPNSITELNFKAPPKKGKITTQQFDFHVTDAQ
VPNHKLRIKSTPKDLGIDTVKQYSGYLDVVDEDKHFFYYFFESRNDPKNDPVILWLNGGP
GCSSLTGLFFELGPSSIDKNLKPVYNPHSWNANASVIFLDQPINVGYSYSSQSVSNTIAA
GKDVYAFLQLFFKNFPEYANLDFHIAGESYAGHYIPAFASEILTHPERNFNLTSVLIGNG
LTDPLVQYEYYEPMACGEGGEPSVLEPEECDGMLNSLPRCLSLIESCYESGSVWSCVPAT
IYCNNGQMGPYQKTGRNVYDIRTMCEGSSLCYSQLEYIDQYLNLPEVKKALGAEVDEYQS
CNFDINRNFMFAGDWMKPYQKNVIDLLEKELPVLIYAGDKDFICNWLGNQAWTNRLEWSG
SKGFTKAPVKSWKVGKNAAGEVKNYKHFTFLRVFGGGHMVPYDQPENALDMVNRWISGDY
KY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hahm MS, Chung BH. (2001) Protein Expression and Purification, 22, 101-107 |
| 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 | pET22b(+) |
| 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 | Dilution |
| Wash Buffer | unknown |
| Solubilization Buffer | 50mM Tris-HCl, 3mM EDTA, 6M guanidinium chloride, pH 8.0 |
| Refolding Buffer | 50mM Tris-HCl, 3mM EDTA, 0.5M NaCl, 10-fold molar ratio of CPY propeptide |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The propeptide was produced separately as a polyhistadine fusion protein cloned into pET22b(+) and expressed in BL-21 E. coli as inclusion bodies. The inclusions were purified and refolded using the same conditions listed here, except solubilization was achieved in 3M guanidinium chloride. The protein was purified by IMAC. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 16.5% |
| Purity | 69% |
| Notes | |