期刊
出版社
ELSEVIER
DOI: 10.1016/j.jchromb.2013.03.024
关键词
Aqueous two-phase systems (ATPS); Proline dehydrogenase (ProDH); Pseudomonas fluorescens; Process integration; Purification; Recovery
资金
- Pasteur Institute of Iran [374]
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 SOS-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. (C) 2013 Elsevier B.V. All rights reserved.
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