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Simultaneous Purification and Refolding of Proteins by Affinity Precipitation and Macro (Affinity Ligand)-Facilitated Three Phase Partitioning (MLFTPP)

  • Ipsita Roy
  • Kalyani Mondal
  • Munishwar N. GuptaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1129)

Abstract

This chapter describes two simple interrelated non-chromatographic methods of protein purification. In the first method, called affinity precipitation, inherent affinity of reversibly soluble–insoluble polymers (also called stimuli-sensitive or smart polymers) is exploited to form an affinity complex in free solution with target protein. The affinity complex is precipitated by a suitable change in the medium. The desired protein is dissociated from the smart polymer. In the second method called macro (affinity ligand)-facilitated three phase partitioning (MLFTPP), the affinity complex is precipitated at an interface between upper t-butanol-rich phase and lower aqueous phase. These three phases are achieved by adding appropriate amounts of ammonium sulfate and t-butanol to the initial crude extract. In the first protocol, sequential MLFTPP is used with two different smart polymers to purify pectinase and cellulase from a single crude preparation. The second protocol illustrates the application of the affinity precipitation in simultaneous purification and refolding of a urea-denatured xylanase.

Key words

Affinity precipitation Alginate Cellulase Chitosan Eudragit Macro (affinity ligand) Pectinase Protein purification Smart polymers Three phase partitioning Xylanase 

Notes

Acknowledgements

We acknowledge financial support provided by the Department of Science and Technology (DST) and Department of Biotechnology (DBT), both Government of India organizations. We thank our other colleagues/collaborators whose work we have extensively referred to. One of us (MNG) would like to especially thank Prof. Bo Mattiasson who way back taught him the art and science of affinity precipitation and provided excellent hospitality at Chemical Centre, Lund University, Sweden, during that learning period.

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Ipsita Roy
    • 1
  • Kalyani Mondal
    • 2
  • Munishwar N. Gupta
    • 3
    Email author
  1. 1.Department of BiotechnologyNational Institute of Pharmaceutical Education and ResearchS.A.S. NagarIndia
  2. 2.Department of Biochemical Engineering and BiotechnologyIndian Institute of TechnologyHauz Khas, New DelhiIndia
  3. 3.Department of ChemistryIndian Institute of TechnologyHauz Khas, New DelhiIndia

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