| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1212964 | Journal of Chromatography B | 2013 | 7 Pages |
•We report a one-step integrated process for recovery and purification of ProDH.•The process combined two-phase partitioning with refolding and reconstitution.•The optimized conditions were 14% (w/w) PEG-1000/12% (w/w) Na2CO3 at pH 8.0.•SDS-PAGE and enzyme assay confirmed purity and renaturation of recombinant enzyme.
The integration of refolding, reconstitution and two-phase partitioning in aqueous two-phase systems (ATPS) which is composed of polyethylene glycol (PEG) and sodium carbonate (Na2CO3) was employed as a novel method for recovery and purification of recombinant Pseudomonas fluorescens proline dehydrogenase (ProDH). To obtain an optimal condition, the influence of different parameters, such as PEG molecular weight (MW), type and concentration of salt, pH, and NaCl addition on the partitioning features of target enzyme was also investigated. Combining the refolding, reconstitution and two-phase partitioning in an optimized ATPS of 14% (w/w) PEG-1000 and 12% (w/w) Na2CO3 at pH 8.0 resulted in a yield of 61.5%, purification factor of 27.0, recovery of 430.7% and specific activity of 600.0 U/mg. The recombinant P. fluorescens enzyme was preferentially partitioned into the top PEG-rich phase. NaCl addition decreased greatly the partition coefficient and recovery of ProDH. In addition, the resulting protein pattern by SDS-PAGE demonstrated the adequacy of presented procedure for enzyme recovery. Overall, our data confirmed that the PEG-1000/Na2CO3 aqueous two-phase partitioning combined with refolding and reconstitution can be used as an efficient integrated process for recovery and purification of recombinant ProDH from inclusion bodies in only one step.
