RFP tags for labeling secretory pathway proteins

Biochem Biophys Res Commun. 2014 May 9;447(3):508-12. doi: 10.1016/j.bbrc.2014.04.013. Epub 2014 Apr 13.

Abstract

Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to an environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.

Keywords: Artificial puncta; Evaluation method; K(d); Red fluorescent proteins; pKa.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • HEK293 Cells
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Lysosomes / metabolism
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Molecular Imaging / methods*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Red Fluorescent Protein
  • Secretory Pathway
  • Staining and Labeling / methods*

Substances

  • Luminescent Proteins
  • Membrane Proteins
  • Recombinant Fusion Proteins