Refolding Record:
| Protein | |
|---|---|
| Protein Name | Receptor-type adenylate cyclase GRESAG 4 |
| Abbreviated Name | GRESAG 4.1 |
| SCOP Family | Adenylyl and guanylyl cyclase catalytic domain |
| Structure Notes | |
| Organism | Trypanosoma brucei |
| UniProt Accession | Q99279 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Cytosolic Domain (residues: 884-1242) |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 380 |
| Molecular Weight | 42483.4 |
| Pi | 5.84 |
| Molecular Weight | 42483.4 |
| Disulphides | 0 |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSHMAERRNNNRAPKEPTDPVTLIFTDIESSTALWAAHPDLMPDAVAAHHRMVRSLIGRYKCYEVKTVGDSFMIASKSPFAAVQLAQELQLCFLHHDWGTNALDDSYREFEEQRAEGECEYTPPTAHMDPEVYSRLWNGLRVRVGIHTGLCDIIRHDEVTKGYDYYGRTPNMAARTESVANGGQVLMTHAAYMSLSAEDRKQIDVTALGDVALRGVSDPVKMYQLNTVPSRNFAALRLDREYFFDEGEDGTTTSTSDHSSSRADVSESGQIIATALQSLLSTFKTAHREKLLLPYCERWRVPLPRKAASEWDDAYCEEVVRRIAVKVGRVADHGADSGSESSSTQGSSSIIIVPFYDMHLQEY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bieger, B., Essen, L. O. (200) Acta Cryst., 56, 359-362 |
| Project Aim | Crystallography |
| Fusion | N-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 | 6h |
| Expression Vector | pET-28a |
| Expression Protocol | Cells were grown in LB medium containing 50microg/ml kanamycin. After induction and incubation cells were harvested and resuspended in 1/100th of the culture volume with PBS, 2mM DTT, 0.2mM PMSF, 1mM EDTA and lysed by two passes through a French Press. DNAse was added to the lysate which was sonified by three continues pulses. Inclusion bodies were then collected by centrifugation(25,000g, 30min). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 595 = 0.5-0.6 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M guanidinium chloride, pH 8.0, |
| Refolding Buffer | A. 20mM TrisHCl, pH 8.0, 0.4M arginine B. 20mM TrisHCl, pH 8.0, 0.5mM DTT |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 18h |
| Redox Agent | DTT |
| Redox Agent Concentration | 0.5mM |
| Refolding Protocol | Inclusion bodies were resupended in solubilisation buffer, clarified and centrifuged(25,000g, 30min). The supernatant was loaded onto a 15ml Ni-NTA-agarose column, pre-equilibrated with 8M urea, 20mM imidazole hydrochloride, pH 8.0. The eluate fractions were then brought to 6M guanidine hydrochloride, 0.1 M DTT, 0.4M arginine hydrochloride, pH 8.0. The adenylate cylase fragments were refolded by a preliminary 1:20 dialysis against refolding buffer A for 12h, followed by further dialysis against refolding buffer B for 6h. |
| Refolding Assay | Crystallography,Mass spectrometry |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.4M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |