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Partitioning behavior of enhanced green fluorescent protein in nickel-chelated affinity-based aqueous two-phase micellar system and its purification from cell lysate

Authors
Journal
Separation and Purification Technology
1383-5866
Publisher
Elsevier
Identifiers
DOI: 10.1016/j.seppur.2014.07.005
Keywords
  • Liquid–Liquid Extraction
  • Aqueous Two-Phase Micellar System
  • Nickel-Chelated Surfactant
  • Green Fluorescent Protein
  • Purification
Disciplines
  • Biology

Abstract

Abstract Aqueous two-phase micellar system (ATPMS), an alternative to chromatography, has been considered as a promising liquid–liquid extraction technique for biomolecule purification. To improve the specificity of ATPMSs, a nickel-chelated surfactant (TX-Ni) has been fabricated. The affinity-based ATPMS formed by Triton X-114 (TX) and TX-Ni was characterized for the purification of recombinant hexahistidine-tagged enhanced green fluorescent protein (EGFP). The stability of EGFP in the ATPMS was first confirmed. Then, the affinity binding of EGFP to TX-Ni was proved by investigation of the partitioning behavior. Thereafter, EGFP was extracted directly from cell lysate by the Ni(II)-chelated ATPMS. It was found that, more impurities were removed to the micelle-poor phase with increasing NaCl concentration, and the increase of TX-Ni gave rise to a recovery of EGFP over 90%. Finally, ethylenediaminetetraacetic acid (EDTA) was used to back-extract EGFP, presenting a total recovery yield of 83% with a purity of 70%. The results indicate that the affinity-based ATPMS is promising for the primary separation of histidine-rich proteins.

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