Refolding Record:
| Protein | |
|---|---|
| Protein Name | Ribonuclease pancreatic |
| Abbreviated Name | RNase A |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | P61823 |
| 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 | 124 |
| Molecular Weight | 13690.3 |
| Pi | 8.63 |
| Molecular Weight | 13690.3 |
| Disulphides | 4 |
| Full Sequence |
KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Raghava S, Barua B, Singh PK, Das M, Madan L, Bhattacharyya S, Bajaj K, Gopal B, Varadarajan R, Gupta MN. (2008) Protein Science, 1, 1 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | E coli |
| Expression Strain | BL21 (DE3) |
| Expression Temp | 37.0 |
| Expression Time | 12 h |
| Expression Vector | pET-22b+ |
| Expression Protocol | The plasmid pET-22b (+) containing the coding region for full length bovine pancreatic RNase A insert was transformed into E. coli BL21 (DE3). A single colony was picked and inoculated into 5 mL LB medium containing 100 µgmL-1 ampicillin. The tube was shaken overnight at 37 °C at 200 rpm. 1% of primary inoculum was transferred into 1 L fresh LB broth (amp+) and grown at 37 °C with vigorous shaking until A600 reached 0.8. IPTG was added to a final concentration of 0.5 mM and the culture was further grown under similar conditions for 12 h. The above procedure was repeated for the transformation of the plasmids pBAD24 containing MBP224D and MBP264D inserts, pET22b containing DLAR, DPTP10D, DPTP69D, DPTP52F and DPTP99A inserts, and pET20b(+) containing (A14E)malETrx insert [showing leaky expression] into E. coli BL21 (DE3). The plasmid pET-28a expressing CD4D12 was transformed into E. coli BL21 (DE3) and 50 µg mL-1 kanamycin was used as the selection marker. In case of MBP224D and MBP264D, induction was carried out by adding L-arabinose (0.2%) and the culture was further grown under similar conditions for 12 h. The plasmid pBAD24 expressing CcdB mutants F17P or M97K was transformed into expression strain E. coli CSH501. Induction was carried out by adding L-arabinose (0.2%) and the culture was further grown under similar conditions for 12 h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.8 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Three phase partitioning |
| Wash Buffer | 50 mM PBS/pH 7.4/1 mM EDTA and further with buffer containing 2M urea |
| Solubilization Buffer | 8 M urea/100 mM DTT |
| Refolding Buffer | 50 mM Tris-HCl, pH 7.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Refolding of solubilized inclusion bodies by three phase partitioning (TPP) Washed inclusion bodies were subjected to solubilization with 8 M urea/100 mM DTT at 25 °C for 3 h. The solubilized sample was saturated with ammonium sulfate (5%, wv-1) and vortexed gently to dissolve the salt. Following this, t-butanol was added in 1:1 (vv-1) ratio of solubilized sample to t-butanol. After incubation for 1 h at 25°C, the mixture was centrifuged [4,000g for 5 min] to facilitate separation of phases. Three phases were formed: the upper organic phase, an interfacial precipitate and lower aqueous phase [see Figure 1]. The aqueous layer was subjected to a second round of TPP. The aqueous phase was saturated with ammonium sulfate [35%, wv-1] followed by addition of t-butanol [in 1:1 (vv-1) ratio of aqueous phase to t-butanol]. Again three phases were formed and collected. The interfacial precipitate was dissolved and dialyzed against 0.1 M Tris-HCl, pH 7.1, containing 0.1 M NaCl in case of RNase A; 50 mM Tris-HCl, pH 7.5 for CcdB mutants; 50 mM Tris-HCl, pH 7.0, containing 150 mM NaCl in case of MBP mutants; 0.1 M phosphate buffer, pH 7.0 in case of Trx mutant; 50 mM Tris-HCl, pH 7.0, containing 150 mM NaCl, 1 mM EDTA in case of PTPs; PBS, pH 7, for CD4D12 [two changes at an interval of 2 h and final dialysis for 6-7 h at 4 °C]. In all the cases the interfacial precipitate from the second TPP was used after dissolution and dialysis for activity measurements. In case of CD4D12 to remove a few higher molecular weight contaminants, the refolded protein solution was subjected to ltrafiltration using a 50 kDa polyethersulfone membrane [PALL Lifesciences]. In the control experiment, solubilized inclusion body samples of the same protein concentration were subjected to dialysis for the same time without TPP. For refolding RNase A, after dialysis the sample was kept for air oxidation in an open vessel at 25 °C for 24 h. |
| Refolding Assay | Protein activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 40-45 mg/l |
| Purity | n/a |
| Notes | n/a |