Refolding Record:
| Protein | |
|---|---|
| Protein Name | Creatine kinase |
| Abbreviated Name | CK |
| SCOP Family | Guanido kinase catalytic domain |
| Structure Notes | |
| Organism | Torpedo californica |
| UniProt Accession | P04414 |
| 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 | 387 |
| Molecular Weight | 42934.0 |
| Pi | 6.40437 |
| Molecular Weight | 42934.0 |
| Disulphides | 0 |
| Full Sequence |
MPFGNTHNKWKLNYSAAEEFPDLSKHNNHMAKALTLDIYKKLRDKETPSGFTLDDIIQTG
VDNPGHPFIMTVGCVAGDEECYEVFKDLFDPVIEDRHGGYKPTDKHKTDLNQENLKGGDD
LDPNYVLSSRVRTGRSIKGIALPPHCSRGERRLVEKLCIDGLATLTGEFQGKYYPLSSMS
DAEQQQLIDDHFLFDKPISPLLLASGMARDWPDGRGIWHNNDKTFLVWVNEEDHLRVISM
QKGGNMKEVFRRFCVGLKKIEDIFVKAGRGFMWNEHLGYVLTCPSNLGTGLRGGVHVKIP
HLCKHEKFSEVLKRTRLQKRGTGGVDTAAVGSIYDISNADRLGFSEVEQVQMVVDGVKLM
VEMEKRLENGKSIDDLMPAQK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Babbitt PC, West BL, Buechter DD, Kuntz ID, Kenyon GL (1990) BIO/TECHNOLOGY, 8, 945-949 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JA221 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pKTCK3F |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris, 8% sucrose, 5% Triton X-100, 50mM EDTA, pH 8.5 |
| Solubilization Buffer | 50 mM Tris, 6M guanidinium chloride, 100mM DTT pH 8.5 |
| Refolding Buffer | 50mM Tris |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 37.0 |
| Protein Concentration | 1 mg/ml |
| Refolding Time | 14h |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The inclusion bodies were washed with 50mM Tris, 8% sucrose, 5% Triton X-100, 50mM EDTA, pH 8.5 in order to remove a proteolytic contaminant that significantly reduces refolding yield. Subsequently, the insoluble protein was solubilized in 50 mM Tris, 6M guanidinium chloride, 100mM DTT pH 8.5 and refolded by 60 fold dilution with rapid mixing at room temperature for 20min-2h. The solution was then stored at 4C overnight. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 0.57mg/L culture |
| Purity | 2 to 3 very faint impurity bands on SDS PAGE. |
| Notes | THE REFOLDING TEMPERATURE WAS ENTERED AS 0 DEGREES BUT WAS CHANGED TO 37 DEGREES - JASON |