Refolding Record:
| Protein | |
|---|---|
| Protein Name | N-carbamoyl-D-amino acid amidohydrolase |
| Abbreviated Name | DCaseH |
| SCOP Family | Carbamilase |
| Structure Notes | |
| Organism | Agrobacterium radiobacter |
| UniProt Accession | Q44185 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Tetramer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 309 |
| Molecular Weight | 34152.1 |
| Pi | 6.00777 |
| Molecular Weight | 34152.1 |
| Disulphides | Unknown |
| Full Sequence |
MTRQMILAVGQQGPIARAETREQVVGRLLDMLTNAASRGVNFIVFPELALTTFFPRWHFT
DEAELDSFYETEMPGPVVRPLFETAAELGIGFNLGYAELVVEGGVKRRFNTSILVDKSGK
IVGKYRKIHLPGHKEYEAYRPFQHLEKRYFEPGDLGFPVYDVDAAKMGMFICNDRRWPET
WRVMGLKGAEIICGGYNTPTHNPPVPQHDHLTSFHHLLSMQAGSYQNGAWSAAAGKVGME
EGCMLLGHSCIVAPTGEIVALTTTLEDEVITAAVDLDRCRELREHIFNFKAHRQPQHYGL
IAEF
|
| Notes | THE UNIPROT ID IS FOR A PROTEIN WITH 100% IDENTITY WITH DCase THOUGH IS FROM Agrobacterium tumefaciens, NOT Agrobacterium radiobacter - JASON |
| Expression | |
|---|---|
| Report | Chen HM, Lin KY, Lu CH (2005) Process Biochemistry, 40, 2135-2141 |
| Project Aim | Folding |
| 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 | 6 hours |
| Expression Vector | pDAH2 |
| Expression Protocol | Enzyme Production. E. coli BL21(DE3) carrying pDAH and pDAH2 were the strains used to produce intact WT DCase and fusion DCaseH enzymes, respectively. Both enzymes were expressed under the control of the bacteriophage T7 promoter. For routine production, a Luria-Bertani (LB) broth containing 30 g/mL kanamycin was inoculated by a single colony and incubated overnight at 37 C. The culture was then diluted 10-fold with the same medium and grown in a shaking flask. When the OD600nm value reached 0.4-0.5, isopropyl--D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.02 mM to induce the protein expression, and the incubation was continued for another 6 h at 37 C before cell harvesting. To investigate the optimal conditions for the enzyme production, cell pellets harvested from 10 mL cultures, which were cultivated with the variation of the IPTG concentration and the cultivation time and temperature after the induction, were suspended in 1 mL of phosphate buffer (PB; 50 mM Na2HPO4, 50 mM NaH2PO4, pH 7) containing 5 mM dithiothreitol (DTT), disrupted by sonication, clarified by centrifugation, and assayed. To solubilize the intracellular insoluble fraction, 1 mL of 8 M urea was added. The cell pellets from 40 mL of the culture were then harvested by centrifugation, resuspended in 5 mL of lysis buffer (0.1 M NaH2PO4, 0.01 M Tris–HCl, 10 mM ?-mercaptoethanol (?-ME), pH 8), sonicated, and centrifuged. The clear supernatant was then mixed with Co-TANOL gels to purify native DCaseH, and the insoluble fraction was first washed twice with lysis buffer and subsequently either refolded as described below or purified with denaturing IMAC. Both native and denaturing IMAC purifications of DCaseH were performed, as previously described |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.4 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | unknown |
| Solubilization Buffer | 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, 10 mM BME, 0.5 mM PMSF, pH 8 |
| Refolding Buffer | 0.1 M NaH2PO4, 0.01 M Tris-HCl, 10 mM BME, 0.5 mM PMSF, pH 8, 10-50% Glycerol |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 40 ug/ml |
| Refolding Time | 9 days |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 10 mM |
| Refolding Protocol | To refold inclusion bodies containing DCaseH directly, the washed inclusion pellets from 40 mL of culture were resuspended in 5 mL of denaturing buffer (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris–HCl, 10 mM ?-ME, 0.5 mM phenyl methyl sulphonyl fluoride (PMSF), pH 8), incubated at room temperature until complete solubilization, clarified by centrifugation, and assayed for protein concentration. The solution was then rapidly diluted at least 20-fold to 20–100 ?g/mL total protein concentrations with various refolding buffers, gently agitated at 4 °C for at least 16 h, and assayed. The refolding buffers were constituted of 0.1 M NaH2PO4, 0.01 M Tris–HCl, 10 mM ?-ME, 0.5 mM PMSF, and different additives at pH 8, including dextran (3%, w/v), glycerol (10?50%, v/v), and PEG (MW = 3350; 10?30%, w/v or 8000; 10?20%, w/v). To investigate the pH effect on refolding, refolding buffers containing 50% glycerol at different pHs (6.5, 7.0, 7.5, and 8.0) were used. After the refolding, DCaseH was purified by native IMAC and subsequently dialyzed, following the procedures as previously described [11]. To refold IMAC-purified denatured DCaseH, denatured DCaseH eluted from Co-TANOL gels was first dialyzed with the denaturing buffer to remove imidazole. After the determination of the protein concentration, the solution was rapidly diluted by about 40-fold to 50 ?g/mL with the refolding buffers containing either 40 or 50% glycerol at pH 8, gently agitated at 4 °C for at least 24 h, and subjected to the enzymic assay and CD analysis. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 10-50% |
| Refolding Yield | 2.54 U/mg |
| Purity | 37% |
| Notes | Optimal results from 50% glycerol for 22 days, though 9 days was only slightly less efficient. |