Biotechnology and Bioprocess Engineering:BBE

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Purification and Characterization of Crystalins by Aqueous Two-Phase Extraction

  • Bermudez, Ondrea (Chemical Engineering Department, University of Missouri-Rolla, Rolla, MO65409, USA) ;
  • Forciniti, Daniel (Chemical Engineering Department, University of Missouri-Rolla, Rolla, MO65409, USA)
  • Published : 2001.12.01
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Abstract

Crystallins are a family of water-soluble proteins that constitute up to 90% of the wa-ter-soluble proteins in mammalian eye lenses, We present in this paper an alternative purification method for these proteins using polyethylene glycol/dextran aqueous two-phase extraction. Un-der the appropriate conditions, we were able to recover the γ-crystallin fraction essentially free of the remaining proteins. High concentrations of salt at a neutral pH maximize the recovery of γ-crystallins in the top phase and minimize the contamination by the other proteins present in the lenses. The proposed protocol decreases the separation time by about 50%. The complex partition behavior observed for these proteins reflects a delicate balance between protein/phase-forming species(various polymers and salts) and protein interactions. This is evidenced, in part, by the role played by the largest proteins in this group as a "pseudo"phase-forming species.

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References

  1. Partition of Cell Particles and Macromolecules. (3rd edition) Albertsson, P. A
  2. Enzyme Microb. Technol v.13 Purification and aqueous two-phase partitioning properties of recombinant Vitreoscilla hemoglobin Hart, R. A;J. E. Bailey
  3. Biotechnol. Tech v.4 Metal affinity extraction of human hemoglobin in an aqueous polyethylene glycol-dodium sulfate two-phase sysem Plunkett, S. D;F. H. Arnold
  4. Methods Enzym v.228 Charge determination by Partitioning Johansson, G
  5. Aqueous Two-Phase Partitioning Zaslavsky, B. Y
  6. Methods Enzymol v.228 Cross-partitioning: Determination of isoelectric point by partitioning Walter, H;D. Forciniti
  7. Proc. Natl. Acad. Sci. USA v.88 Binary-liquid phase separation of lens protein solutions Broide, M. L;C. R. Berland;J. Pande;O.O.Ogun;G. B. Benedek
  8. Proc. Natl. Acad. Sci. USA v.84 Binary liquid phase separation and critical phenomena in a protein/water solution Thomson, J. A;P. Schurtenmberger;G. M. Thurston;G. B. Benedek
  9. Inves. Ophthalmol. Vis. Sci v.19 The effects of glycols, aldegydes, and acrylamide on phase separation and opacification in the calf lens Clark, J. I;G. B.Benedek
  10. Biophys. J v.21 Protein interactions in the calf eye lens: Interactions between β-crystallins are repulsive whereas in γ-crystallins they are atractive Eur Tardieu, A;F. Veretout;B. Krop;C. Slingsby
  11. Eur. J. Biochem v.243 A small angle neutron scattering study of proteins at their isoelectric point Edwards, M;P. Petitt;D. Forciniti
  12. J. Chromatogr v.B743 Cold cataracts: A naturally occurring aqueous two-phase system P. Petit; D. Forciniti
  13. Preparation of aqueous two-phase systems, Aqueous Two-Phase Systems-Methods and Protocols Forciniti, D;R. Hatti-Kaul(ed)
  14. Partitioning in Aqueous Two-Phase Systems Partitioning of Proteins. Johansson, G;H.Walter;D. E. Brooks;D. Fisher(eds)
  15. Heme, Chlorophyll and Bilins: Methods and Protocols Hemoproteins purification and characterization by using aqueous two-phase systems Forciniti, D;A. G. Smith;M. Witty(eds)
  16. Biotechnol. Bioeng v.38 Protein partitioning at the isoelectric point: Effect of polymer concentration and polymer molecular weight. Forciniti, D;C.K. Hall;M. -R. Kula
  17. Chem. Eng. Sci v.47 Protein partitioning: Effect of pH and polymer molecular weight Forciniti, D;C. K. Hall;M.-R. Kula