Refolding Record:
| Protein | |
|---|---|
| Protein Name | Exopolyphosphatase |
| Abbreviated Name | Exopolyphosphatase |
| SCOP Family | Ppx/GppA phosphatase |
| Structure Notes | |
| Organism | Trypanosoma brucei |
| UniProt Accession | Q7Z032 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 390 |
| Molecular Weight | 43817.2 |
| Pi | 5.7 |
| Molecular Weight | 43817.2 |
| Disulphides | Unknown |
| Full Sequence |
MTAVVNEFLK RGVRALAARQ DSVLVMGNEG GDMDTVIGSI FLAMYLEKRD VFGVGSYVPV LNFEKDDLPL RQDVVKLLSR HNVSTDSIYS VKQSGNGVDF LDLHQMKLPI VLYDHNKLSP EQVYLGERIV GVVDHHEDEQ LYVDQTKCLR RICKTGSACT LVAELFNEAG LEVPCPELLL APIVVDTVNF EPSQKRVTER DILASRLLVG RDDCGDYLTG MFKELMAWKN DIHCLTVPQH LRRDYKNFEF PFISAENKIL RVGISSISCR SDELLSVHGV SSVSSNCIEF IKQRSIDMLL LTFAGERTPG SYCRQLGVVA KDEGLAALQA YCKKSPSGLD FTLLEDVTVN EWAFSIYELS DPSVSRKKLV PSLTSFLSVW NNL EHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lemercier G, Bakalara N, Santarelli X. (2003) J Chromatography B, 786, 305-309 |
| Project Aim | Undefined |
| 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 |
| Expression Temp | 25.0 |
| Expression Time | 12-16h |
| Expression Vector | pET23a |
| Expression Protocol | The E. coli BL21 strain transformed with the plasmid pET 23a containing the exopolyphosphatase His-tag construction was cultured in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.5) with ampicillin (100 μg/ml). The volume of the culture medium was 80-fold higher than the inoculated volume. The culture was grown at 37degC to 2×108 cells/ml (A600nm=0.7). Then IPTG was added to a final concentration of 0.5 mM to a culture stabilized and incubated overnight at room temperature. These culture conditions did not help for soluble protein production but they allowed a high production of a non-proteolysed protein. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.7 |
| Cell Disruption Method | Freeze/Thaw+Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | 5 mM Imidazole, 0.5 M NaCl, 20 mM TrisHCl, 2-4M urea, 1% Triton X-100 pH 7.9 |
| Solubilization Buffer | 5 mM Imidazole, 0.5 M NaCl, 20 mM TrisHCl, 6 M urea plus 1% Triton X-100 pH 7.9 |
| Refolding Buffer | 5 mM imidazole, 0.5 M NaCl, 20 mM TrisHCl, 1% Triton X-100 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.9 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 14h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The buffer used during equilibration was 5 mM imidazole, 0.5 M NaCl, 20 mM TrisHCl, pH 7.9, 6 M urea, Triton X-100 UV. Refolding of the bound protein was performed on-column by the use of a linear gradient from 6 M to 0 M urea, starting with the equilibration buffer and finishing with a buffer containing 5 mM imidazole, 0.5 M NaCl, 20 mM TrisHCl, pH 7.9, Triton X-100 UV reduced 1%. The refolded protein was eluted with a buffer containing 1 M imidazole, 0.5 M NaCl, 20 mM TrisHCl, pH 7.9, Triton X-100 |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 51% |
| Purity | |
| Notes | |