Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

Patrick J. MacDonald, Yan Chen, Joachim D. Mueller

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42 Scopus citations


Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37 °C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction.

Original languageEnglish (US)
Pages (from-to)291-298
Number of pages8
JournalAnalytical Biochemistry
Issue number1
StatePublished - Feb 1 2012

Bibliographical note

Funding Information:
The authors thank Vincent Noireaux (University of Minnesota) for many useful discussions and suggestions. This work was supported by grants from the National Institutes of Health ( GM64589 ) and the National Science Foundation ( PHY 0346782 ).


  • Brightness analysis
  • Chromophore formation
  • EGFP
  • NTF2
  • Temperature
  • mCherry


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