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Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics

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Abstract

A number of studies have been conducted to improve chromophore maturation, folding kinetics, thermostability, and other traits of green fluorescent protein (GFP). However, no specific work aimed at improving the thermostability of the yellow fluorescent protein (YFP) and of the pH-sensitive, yet thermostable color variants of GFP has so far been done. The protein variants reported in this study were improved through rational multiple site-directed mutagenesis of GFP (ASV) by introducing up to ten point mutations including the mutations near and at the chromophore region. Therefore, we report the development and characterization of fast folder and thermo-tolerant green variant (FF-GFP), and a fast folder thermostable yellow fluorescent protein (FFTS-YFP) endowed with remarkably improved thermostability and folding kinetics. We demonstrate that the fluorescence intensity of this yellow variant is not affected by heating at 75 °C. Moreover, we have developed a pH-unresponsive cyan variant AcS-CFP, which has potential use as part of in vivo imaging irrespective of intracellular pH. The combined improved properties make these fluorescent variants ideal tools to study protein expression and function under different pH environments, in mesophiles and thermophiles. Furthermore, coupling of the FFTS-YFP and AcS-CFP could potentially serve as an ideal tool to perform functional analysis of live cells by multicolor labeling.

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Acknowledgments

The authors would like to thank Dr. Mara Giangrossi of the University of Camerino for kindly providing plasmid pGFP (ASV), Prof. M. Pillay of Vaal University of Technology (Johannesburg-Vanderjiblpark, South Africa) who made it possible to manage the issues of research activity of N.A., and Dr. Stoyan Stoychev of Council for Scientific and Industrial Research (CSIR) (Pretoria, South Africa) for technical assistance during a brief research stay of N.A. at the CSIR. The cost of the research was covered by University of Camerino (Camerino, Italy) and partly by Vaal University of Technology (Johannesburg-Vanderjiblpark, SouthAfrica).

Conflict of interests

The authors declare that there is no financial or any other conflict of interests.

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Authors and Affiliations

  1. Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032, Camerino, Italy

    Naser Aliye, Attilio Fabbretti & Roberto Spurio

  2. Department of Biotechnology, Vaal University of Technology, Private Bag X021 Vanderbijlpark, 1900 Andries Potgieter Boulevard, Johannesburg, Vanderbijlpark, South Africa

    Naser Aliye

  3. Laboratory of Biochemistry, School of Pharmacy, University of Camerino, 62032, Camerino, Italy

    Giulio Lupidi

  4. Council for Scientific and Industrial Research (CSIR), BIOSCIENCES, Meiring Naude Road, Brummeria, Pretoria, 0184, l, South Africa

    Tsepo Tsekoa

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  1. Naser Aliye
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  2. Attilio Fabbretti
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  3. Giulio Lupidi
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  4. Tsepo Tsekoa
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  5. Roberto Spurio
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Correspondence to Roberto Spurio.

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Aliye, N., Fabbretti, A., Lupidi, G. et al. Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics. Appl Microbiol Biotechnol 99, 1205–1216 (2015). https://doi.org/10.1007/s00253-014-5975-1

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  • DOI: https://doi.org/10.1007/s00253-014-5975-1

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