A rapid and efficient protocol to purify biologically active recombinant proteins from mammalian cells

Demian Cazalla, Jeremy R Sanford, Javier F Cáceres

Research output: Contribution to journalArticlepeer-review

Abstract

Here, we describe a simple and efficient method for the expression and purification of active recombinant proteins in mammalian cells. This method uses the expression of T7 epitope-tagged proteins in transiently transfected 293T cells grown in monolayer, followed by anti-T7-agarose affinity chromatography. This procedure yields approximately between 75 and 100 microg of biologically active protein/150 cm(2) flask that can be used for biochemical studies. We have tested this protocol for the expression of the prototype SR protein, SF2/ASF, which is a member of the SR protein family with a role in constitutive and alternative splicing. We show that SF2/ASF purified using this protocol is able to complement an S100 HeLa extract, demonstrating that is biologically active. Moreover, expression of a novel SR-related protein that it is required for the second step of pre-mRNA splicing also rendered an active protein. In summary, we present a protocol based on transient transfection of mammalian cells that results in easy purification of significant amounts of biologically active proteins.
Original languageEnglish
Pages (from-to)54-8
Number of pages5
JournalProtein Expression and Purification
Volume42
Issue number1
DOIs
Publication statusPublished - Jul 2005

Keywords

  • Alternative Splicing
  • Animals
  • Antibodies
  • Bacteriophage T7
  • Capsid Proteins
  • Cell Line
  • Chromatography, Affinity
  • Gene Expression
  • HeLa Cells
  • Humans
  • Nuclear Proteins
  • Plasmids
  • RNA-Binding Proteins
  • Recombinant Proteins

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