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Purification of Recombinant Proteins with a Multifunctional GFP Tag

  • Takashi MurayamaEmail author
  • Takuya Kobayashi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1177)

Abstract

Green fluorescent protein (GFP) is the most widespread fluorescent reporter for cellular localization and interaction of proteins. Because GFP itself is not the protein purification tag, protein purification is generally carried out with the aid of additional affinity tags. We have recently engineered a “multifunctional GFP” (mfGFP), a variant of enhanced GFP (EGFP), in which multiple affinity tags are inserted in tandem into an internal loop of EGFP. The mfGFP can be used as a fluorescent reporter and an affinity tag, and is compatible with various expression systems in prokaryotic and eukaryotic cells. Herein, we describe detailed procedures for the expression and purification of mfGFP fusion proteins in mammalian cells. A method for tandem affinity purification using two different tags within mfGFP is also described.

Key words

Affinity tag Green fluorescent protein (GFP) Immobilized metal affinity chromatography (IMAC) Multifunctional GFP (mfGFP) Protein complex Protein purification Streptavidin binding peptide (SBP) 

Notes

Acknowledgments

This work was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science and the Japan Science and Technology Agency (SENTAN).

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Cellular and Molecular PharmacologyJuntendo University Graduate School of MedicineTokyoJapan

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