caa a22 4500 605501874 CHVBK 20210128100603.0 cr unu---uuuuu 210128e20150201xx s 000 0 eng 10.1007/s00253-014-5975-1 doi (NATIONALLICENCE)springer-10.1007/s00253-014-5975-1 Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics [Elektronische Daten] [Naser Aliye, Attilio Fabbretti, Giulio Lupidi, Tsepo Tsekoa, Roberto Spurio] 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. Springer-Verlag Berlin Heidelberg, 2014 Thermostability nationallicence pH-sensitivity nationallicence Protein engineering nationallicence Cell labeling nationallicence Aliye Naser Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032, Camerino, Italy aut Fabbretti Attilio Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032, Camerino, Italy aut Lupidi Giulio Laboratory of Biochemistry, School of Pharmacy, University of Camerino, 62032, Camerino, Italy aut Tsekoa Tsepo Council for Scientific and Industrial Research (CSIR), BIOSCIENCES, Meiring Naude Road, Brummeria, l, 0184, Pretoria, South Africa aut Spurio Roberto Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032, Camerino, Italy aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/3(2015-02-01), 1205-1216 0175-7598 99:3<1205 2015 99 253 https://doi.org/10.1007/s00253-014-5975-1 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-5975-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Aliye Naser Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032, Camerino, Italy aut NATIONALLICENCE 700 1- Fabbretti Attilio Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032, Camerino, Italy aut NATIONALLICENCE 700 1- Lupidi Giulio Laboratory of Biochemistry, School of Pharmacy, University of Camerino, 62032, Camerino, Italy aut NATIONALLICENCE 700 1- Tsekoa Tsepo Council for Scientific and Industrial Research (CSIR), BIOSCIENCES, Meiring Naude Road, Brummeria, l, 0184, Pretoria, South Africa aut NATIONALLICENCE 700 1- Spurio Roberto Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032, Camerino, Italy aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/3(2015-02-01), 1205-1216 0175-7598 99:3<1205 2015 99 253