Refolding Record:
| Protein | |
|---|---|
| Protein Name | RNA-editing complex protein MP42 |
| Abbreviated Name | TbMP42 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Trypanosoma brucei |
| UniProt Accession | Q95W13 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 404 |
| Molecular Weight | 43534.8 |
| Pi | 7.38 |
| Molecular Weight | 43534.8 |
| Disulphides | 0 |
| Full Sequence |
MKRVTSHISRFLPLVLSQRGLSTYASPHVSSMRYYGATKCLLASTPDTPSFQCGECGKAF
RLINALNHHIMTKHAGKAKAMMNKGGKLEEVNPEEIKNKPQGAMSQPTSPPPSSSTSGTE
AASTSPTHSSFPGIPFSPVGGIGLVGTPVGAASHVVNTTTTAANSASGNALSDENADKKT
FVCTICQKTFRLEAALQHHYQAKHNMEMPTSSSSSGGASAQPVLQGGATTAGVGSVGFSH
TEEETGRSAMGTQYVHSQETILPQAPQYHLDVAPNAPEEGEVAAHWRCVNHCVMLGVVQN
IQEGFVFEDKVLQFTLITDFEGPSPGDPDKDFHTVRVFDSDYSSRVKEQLRDGEWFLVTG
RLRMVPQYDGSMRKYYHYPVIQVHPGCGSVLKVGGSRSHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Brecht, M., Niemann, M., Schluter, E., Muller, U. F., Stuart, K., Goringer, H. U. (2005) Mol Cell, 17, 621-630 |
| Project Aim | Functional Studies |
| Fusion | C-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 | 3h |
| Expression Vector | pQE60 |
| Expression Protocol | Cells were grown in 1L culture, induced and incubated. Cells were then harvested, and lysed in lysis buffer (10mM TrisHCl, pH 8.0, 0.1M NaH2PO4, 8M urea). Lysates were loaded onto a Ni-chelating column and bound protein eluted using a pH step gradient (pH 6.3, pH 5.9, pH 3.0). Fractions containing rTbMP42 were then purified by anion exchange chromatography under denaturing conditions (8M Urea). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5-0.6 |
| Cell Disruption Method | Chemical |
| Lytic Agent | Chemicals |
| Pre-Refolding Purification | Metal affinity chromatography/ion exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea |
| Refolding Buffer | 20 mM HEPES, pH 7.5, 30 mM KCL, 10 mM Mg(OAc)2, 5 mM CaCl2, 1 mM ZnSO4 |
| Pre-Refolding Purification | Metal affinity chromatography/ion exchange chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Refolding was preformed, after purification steps, by dialysis against refolding buffer. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 0.12mg/ml |
| Purity | n/a |
| Notes | n/a |