Refolding Record:
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 3 |
| Molecular Weight | 203.2 |
| Pi | 5.52 |
| Molecular Weight | 203.2 |
| Disulphides | 3 |
| Full Sequence |
n/a
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Boyle DM, Johnson GV, Heeren RA, Shell RE, Banerjee A, Gustafson ME. (2008) Biotechnol Appl Biochem., 49, 73-83 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DH5a |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | n/a |
| Expression Protocol | The construction of the plasmid expression vector for PMP-1a and transformation into E. coli have been described elsewhere [19]. IBs were obtained from high-density fermentations following initial growth in shake flasks incubated at 37 °C and shaken at 200 rev./min for 7.5 h to reach a D550 (attenuance) of 5.5. Approx. 500 ml of the primary seed inoculum was transferred to a 10-litre fermenter containing M9 medium [19a]+2% casamino acids with glucose concentrations maintained between 2 and 6 g/l. The culture was continued at 37 °C with aeration and agitation. The pH of the culture was maintained at 7.0 by the addition of ammonium hydroxide, which also served as a nitrogen source. The fermentation was grown for an additional 4 h following induction with nalidixic acid at a D550 value of 13.5. The fermentation broth was cooled to 15 °C and transferred to a harvest tank. The broth was centrifuged to collect the cells, and the resulting cells were washed once by resuspension and centrifugation at 10 °C in 10 mM Tris/EDTA (pH 8.0) followed by homogenization using a microfluidizer (3000 lbf/in2; 1 lbf/in2=6.9 kPa; Microfluidics). The IB slurry thus obtained was collected by centrifugation and washed once with chilled, purified water. The final slurry was resuspended in purified water to 8–10% (w/v) solids and was stored at –70 °C. |
| Method of Induction | Nalidixic Acid |
| Cell Density at Induction | OD 13.5 = 550 |
| Cell Disruption Method | Microfluidizer |
| Lytic Agent | None |
| Pre-Refolding Purification | RP-HPLC |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | chilled,purified watter |
| Solubilization Buffer | 25 mM Tris buffer (pH 9.7) using 6 M deionized urea and up to 0.6% SDS |
| Refolding Buffer | 25 mM Tris buffer (pH 9.7) using 6 M deionized urea and up to 0.6% SDS or by the addition of 1 mM cysteine |
| Pre-Refolding Purification | RP-HPLC |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 1 h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 1 mM |
| Refolding Protocol | Refolding of PMP-1a IBs were dissolved to approx. 10 mg of protein/ml between 4 and 12 °C in 25 mM Tris buffer (pH 9.7) using 6 M deionized urea and up to 0.6% SDS (SDS was precipitated at levels above 0.6% at 4 °C, so this was considered the practical upper limit), or using other detergents with or without organic modifiers as indicated for the initial refolding attempts. Dissolved IB protein solutions were then diluted to the indicated detergent or urea (≤1.5 M) concentrations to control final refold protein concentrations between 0.2 and 2 mg/ml depending on the experiment. For the initial detergent-comparison studies, two concentrations were evaluated: 0.2 and 0.5%. After solubilization, refolds were initiated by dilution or by the addition of 1 mM cysteine after 1 h or in combination as described above. Untreated samples served as the control. Each detergent was dissolved in 100 mM Tris/HCl, pH 9.7, with a protein concentration of 0.2 mg of PMP-1a/ml determined by quantitative RP-HPLC. Samples were taken at dissolution (t=0), 1 h after cysteine addition and for timed points after that up to 72 h, held at 2–8 °C in sample trays and typically assayed immediately by RP-HPLC and both SDS and non-SDS SEC–HPLC analyses. Although the effect of freeze–thaw on protein oxidation or aggregation was not studied explicitly, samples were occasionally frozen before analysis without unusual results or structural artefacts being observed. At 1–24 h following IB dissolution, catalytic amounts (0.7–5 mM) of the thiol-group-containing reagents cysteine, DTT and/or cystine were added as indicated and refolding was allowed to proceed for 15 h. |
| Refolding Assay | RP-HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |