Refolding Record:
| Protein | |
|---|---|
| Protein Name | MAP kinase phosphatase 3 |
| Abbreviated Name | MKP3 |
| SCOP Family | Dual specificity phosphatase-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q16828 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 381 |
| Molecular Weight | 42319.7 |
| Pi | 4.75007 |
| Molecular Weight | 42319.7 |
| Disulphides | 0 |
| Full Sequence |
MIDTLRPVPFASEMAISKTVAWLNEQLELGNERLLLMDCRPQELYESSHIESAINVAIPGIMLRRLQKGNLPVRALFTRGEDRDRFTRRCGTDTVVLYDESSSDWNENTGGESVLGLLLKKLKDEGCRAFYLEGGFSKFQAEFSLHCETNLDGSCSSSSPPLPVLGLGGLRISSDSSSDIESDLDRDPNSATDSDGSPLSNSQPSFPVEILPFLYLGCAKDSTNLDVLEEFGIKYILNVTPNLPNLFENAGEFKYKQIPISDHWSQNLSQFFPEAISFIDEARGKNCGVLVHCLAGISRSVTVTVAYLMQKLNLSMNDAYDIVKMKKSNISPNFNFMGQLLDFERTLGLSSPCDNRVPAQQLYFTTPSNQNVYQVDSLQST
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Mark JK, Smith S, Hefford MA (2007) Protein Expression and Purification, 999, 999 |
| Project Aim | Functional Studies,Drug Studies |
| Fusion | N-terminal hexahistidine tag + thrombin cleavage site |
| 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 | 1.5-2 hr |
| Expression Vector | pET-15b |
| Expression Protocol | An MKP3-producing BL21(DE3)/MKP3 starter culture was established overnight in a 50 mL conical tube containing 12 mL LB broth and 50 microg/mL carbenicillin. The next morning, 10 mL of the culture was added to 1 L of 50 microg/mL carbenicillin supplemented, Luria-Bertani (LB) broth in a 2.8 L Fernbach flask. The production culture was grown with shaking (150 RPM) at 37 °C until an OD600 between 0.4 and 0.5 was reached, whereupon expression of MKP3 was induced by addition of 1 mM IPTG (final concentration). The cells were then allowed to grow for a further 2 h before being harvested by centrifugation (8000 g for 30 min). Cell pellets were stored frozen at -80 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | TBS |
| Solubilization Buffer | TBS containing 6 M guanidine–HCl (pH 7.6) |
| Refolding Buffer | 0.6 M Guanidine, 0.8 M Arginine, 5 mM DTT |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.6 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 1 mg/mL |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a,5 mM,5 mM |
| Refolding Protocol | Refolding conditions were optimized using a 96-well plate format (round-bottom, Corning). Purified protein solutions at 1 mg/mL were rapidly diluted 1:20 into 200 ?L of cold refolding buffers and left overnight at 4 °C to equilibrate. The refolding buffers tested consisted of a TBS-base with modifications to guanidine and arginine concentration. The nine formulations were tested were: #1. 0.3 M Guanidine, 0 M Arginine, 5 mM DTT #2. 0.3 M Guanidine, 0.4 M Arginine, 5 mM DTT #3. 0.3 M Guanidine, 0.8 M Arginine, 5 mM DTT #4. 0.6 M Guanidine, 0 M Arginine, 5 mM DTT #5. 0.6 M Guanidine, 0.4 M Arginine, 5 mM DTT #6. 0.6 M Guanidine, 0.8 M Arginine, 5 mM DTT #7. 0.9 M Guanidine, 0 M Arginine, 5 mM DTT #8. 0.9 M Guanidine, 0.4 M Arginine, 5 mM DTT #9. 0.9 M Guanidine, 0.8 M Arginine, 5 mM DTT Additionally, gradual dialysis was performed as an alternative to dilution-based refolding. Dialysis was performed by first adjusting 6 M guanidine-denatured MKP3 to 0.25 mg/mL and then exchanging the sample into TBS (pH 7.6) containing 1 mM EDTA, 5 mM DTT, and 8 M urea. The gradual removal of urea was completed by a half-wise substitution of dialysis buffer every 4 h using fresh TBS containing 1 mM EDTA and 5 mM DTT at pH 7.6. |
| Refolding Assay | Far-UV Circular Dichroism,Enzyme activity,Western Blot,Mass spectrometry |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.8 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | Due to the problems associated with the residual arginine and guanidine, refolding of MKP3 by dialysis was also tested as an alternative refolding method. MKP3 refolded by this method yielded a comparable specific activity at 11.6 microM NP product/min/mg in the presence of DMSO. While the dilution-based refolding method was a simpler, less time consuming technique for renaturation, the need to perform a final dialysis step to remove residual guanidine and arginine eliminated the benefits of the method. Therefore, stepwise dialysis of the 6 M guanidine-solubilized MKP3 into TBS was adopted for refolding subsequent samples of MKP3. |