Refolding Record:
| Protein | |
|---|---|
| Protein Name | Protein Kinase C |
| Abbreviated Name | PKC |
| SCOP Family | Protein kinase cysteine-rich domain (cys2, phorbol-binding domain) |
| Structure Notes | |
| Organism | Rat |
| UniProt Accession | P63319 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 697 |
| Molecular Weight | 78357.7 |
| Pi | 7.26965 |
| Molecular Weight | 78357.7 |
| Disulphides | 0 |
| Full Sequence |
MAGLGPGGGDSEGGPRPLFCRKGALRQKVVHEVKSHKFTARFFKQPTFCSHCTDFIWGIGKQGLQCQVCSFVVHRRCHEFVTFECPGAGKGPQTDDPRNKHKFRLHSYSSPTFCDHCGSLLYGLVHQGMKCSCCEMNVHRRCVRSVPSLCGVDHTERRGRLQLEIRAPTSDEIHITVGEARNLIPMDPNGLSDPYVKLKLIPDPRNLTKQKTKTVKATLNPVWNETFVFNLKPGDVERRLSVEVWDWDRTSRNDFMGAMSFGVSELLKAPVDGWYKLLNQEEGEYYNVPVADADNCSLLQKFEACNYPLELYERVRMGPSSSPIPSPSPSPTDSKRCFFGASPGRLHISDFSFLMVLGKGSFGKVMLAERRGSDELYAIKILKKDVIVQDDDVDCTLVEKRVLALGGRGPGGRPHFLTQLHSTFQTPDRLYFVMEYVTGGDLMYHIQQLGKFKEPHAAFYAAEIAIGLFFLHNQGIIYRDLKLDNVMLDAEGHIKITDFGMCKENVFPGSTTRTFCGTPDYIAPEIIAYQPYGKSVDWWSFGVLLYEMLAGQPPFDGEDEEELFQAIMEQTVTYPKSLSREAVAICKGFLTKHPGKRLGSGPDGEPTIRAHGFFRWIDWERLERLEIAPPFRPRPCGRSGENFDKFFTRAAPALTPPDRLVLASIDQADFQGFTYVNPDFVHPDARSPTSPVPVPVM
|
| Notes | C1 domains of PKCs (PKC-alpha and PKC-gamma) PKC-alpha C1 domains. C1a - HKFIARFFKQPTFCSHCTDFIWGFGKQGFQCQVCCFVVHKRCHEFVTFSC Number of amino acids: 50 Molecular weight: 5968.0 Theoretical pI: 8.82 C1b - HKFKIHTYGSPTFCDHCGSLLYGLIHQGMKCDTCDMNVHKQCVINVPSLC Number of amino acids: 50 Molecular weight: 5624.5 Theoretical pI: 7.81 PKC-gamma C1 domains C1a HKFTARFFKQPTFCSHCTDFIWGIGKQGLQCQVCSFVVHRRCHEFVTFEC Number of amino acids: 50 Molecular weight: 5941.9 Theoretical pI: 8.68 C1b HKFRLHSYSSPTFCDHCGSLLYCLVHQGMKCSCCEMNVHRRCVRSVPSLC Number of amino acids: 50 Molecular weight: 5760.7 Theoretical pI: 8.74 |
| Expression | |
|---|---|
| Report | Ananthanarayanan, B., Stahelin, R.V., Digman, M.A., Cho, W. (2003) J Biol Chem, 278, 46886-46894 |
| Project Aim | Structure-Function |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pVL1392 |
| Expression Protocol | Not stated |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Other |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50 mM TrisHCl, ph 7.4, 50mM NaCl, 0.4 %(v/v) Triton X-100 and 1 mM phenylmethylsulfonyl fluoride |
| Solubilization Buffer | 50 mM TrisHCl, pH 8.0, 50 mM NaCl, 8 M urea, 0.5 mM dithiothreitol |
| Refolding Buffer | 50 mM TrisHCl, pH 8.0 containing, 50 mM NaCl, 1.5 M Urea, 50 microM ZnSO4, and 0.5 mM dithiothreitol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | C1 domains 1 L of LB medium containing 100 microgram/ml ampicillin, was inoculated with overnight culture. Cells were grown until they reached an OD600 of 0.8-1.4 before being induced with 0.5 mM ITPG. After 4h cells were harvested via centrifugation (50 000g, 25 min, 4degC). Cells were resuspending in 20 ml lysis buffer (same composition as the wash buffer) before sonication. Supernatant containing the soluble, C1B domains of PKC-alpha and PKC-beta was collected via centrifugation (50 000g, 25 min, 4degC). Inclusion body was resuspended in wash buffer, and recentrifuged (50 000g, 25min, 4degC). Washed inclusion body was resuspended in 10 ml of solubilisation buffer before insoluble matter was removed by centrifugation (100 000g for 15 min) and supernatant dialyses against refolding buffer and then against 50 mM TrisHCl pH 8.0. Refolded C1 domain was purified using a nickel-nitrilotriacetic acid column. |
| Refolding Assay | Enzyme activity,enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | C1 domains of PKCs (PKC-alpha and PKC-gamma) PKC-alpha C1 domains. C1a - HKFIARFFKQPTFCSHCTDFIWGFGKQGFQCQVCCFVVHKRCHEFVTFSC Number of amino acids: 50 Molecular weight: 5968.0 Theoretical pI: 8.82 C1b - HKFKIHTYGSPTFCDHCGSLLYGLIHQGMKCDTCDMNVHKQCVINVPSLC Number of amino acids: 50 Molecular weight: 5624.5 Theoretical pI: 7.81 PKC-gamma C1 domains C1a HKFTARFFKQPTFCSHCTDFIWGIGKQGLQCQVCSFVVHRRCHEFVTFEC Number of amino acids: 50 Molecular weight: 5941.9 Theoretical pI: 8.68 C1b HKFRLHSYSSPTFCDHCGSLLYCLVHQGMKCSCCEMNVHRRCVRSVPSLC Number of amino acids: 50 Molecular weight: 5760.7 Theoretical pI: 8.74 |