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

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• 作者：Naser Aliye ; Attilio Fabbretti ; Giulio Lupidi…
• 关键词：Thermostability ; pH ; sensitivity ; Protein engineering ; Cell labeling
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2015
• 出版时间：February 2015
• 年：2015
• 卷：99
• 期：3
• 页码：1205-1216
• 全文大小：1,972 KB
• 参考文献：1. Andersen JB, Sternber C, Poulsen LK, Bjorn SP, Givskov M, Molin S (1998) New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol 64:2240-246
  2. Bizzarri R, Nifosi R, Abbruzzetti S, Rocchia W, Guidi S, Arosio D, Garau G, Campanini B, Grandi E, Ricci F, Viappiani C, Beltram F (2007) Green fluorescent protein ground states: the influence of a second protonation site near the chromophore. Biochemistry 46:5494-504 CrossRef
  3. Bizzarri R, Serresi M, Luin S, Beltram F (2009) Green fluorescent protein based pH indicators for in vivo use: a review. Ann Bioanal Chem 393:1107-122 CrossRef
  4. Cava F, de Pedro MA, Blas-Galindo E, Waldo GS, Westblade LF, Berenguer J (2008) Expression and use of superfolder green fluorescent protein at high temperatures in vivo: a tool to study extreme thermophile biology. Environ Microbiol 10:605-3 CrossRef
  5. Cormack BP, Valdivia RH, Falkow S (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173:33- CrossRef
  6. Crameri A, Whitehorn EA, Tate E, Stemmer WP (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol 14:315-19 CrossRef
  7. Cubitt AB, Woolenweber LA, Heim R (1999) Understanding structure-function relationships in the / Aequorea victoria green fluorescent protein. Methods Cell Biol 58:19-0 CrossRef
  8. Erijman A, Dantes A, Bernheim R, Shifman JM, Peleg Y (2011) Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering. J Struct Biol 175:171- CrossRef
  9. Geldner N, Denervaud-Tendon V, Hyman DL, Mayer U, Stierhof YD, Chory J (2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J 59:169-78 CrossRef
  10. Heim R, Prasher DC, Tsien RY (1994) Wavelength mutations and posttranslational auto oxidation of green fluorescent protein. Proc Natl Acad Sci U S A 91:12501-2504 CrossRef
  11. Heim R, Cubitt A, Tsien R (1995) Improved green fluorescence. Nature 373:663-4 CrossRef
  12. Hunte C, Screpanti E, Venturi M, Rimon A, Padan E, Michel H (2005) Structure of a Na+/H-?antiporter and insights into mechanism of action and regulation by pH. Nature 435:1197-202 CrossRef
  13. Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of / Escherichia coli with plasmids. Gene 96:23-8 CrossRef
  14. Jayaraman S, Haggie P, Wachter RM, Remington SJ, Verkman AS (2000) Mechanism and cellular applications of a green fluorescent protein-based halide sensor. J Biol Chem 275:6047-050 CrossRef
  15. Kiss C, Temirov J, Chasteen L, Waldo GS, Bradbury ARM (2009) Directed evolution of an extremely stable fluorescent protein. Protein Eng Des Sel 22:313-23 protein/gzp006" target="_blank" title="It opens in new window">CrossRef
  16. Kneen M, Farinas J, Li Y, Verkman AS (1998) Green fluorescent protein as a non-invasive intracellular pH indicator. Biophys J 74:1591-599 CrossRef
  17. Llopis J, McCaffery JM, Miyawaki A, Farquhar MG, Tsien RY (1998) Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc Natl Acad Sci USA 95:6803-8
• 作者单位：Naser Aliye (1) (2)
  Attilio Fabbretti (1)
  Giulio Lupidi (3)
  Tsepo Tsekoa (4)
  Roberto Spurio (1)
  
  1. Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032, Camerino, Italy
  2. Department of Biotechnology, Vaal University of Technology, Private Bag X021 Vanderbijlpark, 1900 Andries Potgieter Boulevard, Johannesburg, Vanderbijlpark, South Africa
  3. Laboratory of Biochemistry, School of Pharmacy, University of Camerino, 62032, Camerino, Italy
  4. Council for Scientific and Industrial Research (CSIR), BIOSCIENCES, Meiring Naude Road, Brummeria, Pretoria, 0184, l, South Africa
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

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.