pH Sensor Using Superfolder Green Fluorescent Protein for Intracellular Studies
Kumar, Kumaragurubaran (2014)
Master's Degree Programme in Science and Bioengineering
Luonnontieteiden tiedekunta - Faculty of Natural Sciences
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The aim of this study is to create novel pH reporting probe for use in intracellular studies of thermophiles using superfolder green fluorescent protein. The strains were screened based on ratiometric spectral characteristics. For comparison of results, we have used deGFP mutant which is the best fluorescent protein based ratiometric probe currently available. The work was conducted in different steps including site directed mutagenesis, transformation and cloning, protein expression and spectral measurements. Mutations were created using overlap extension PCR method. We have demonstrated the influence of mutations T203C, H148G and C48S on both expression and spectral characteristics of “superfolder” GFP. sfGFPp1 with mutation T203C is selected as best strain from our study based on ratiometric spectral property. The linear emission response of sfGFPp1 at physiological pH range is very significant. To the best of our knowledge, except sfGFP, all the fluorescent proteins reported are unstable at high temperatures. This study demonstrated the stability of sfGFP and the newly developed variant sfGFPp1 at 70°C while retaining its pH sensitivity making it the only fluorescent protein based pH sensor for thermophilic conditions. . In vitro emission of sfGFPp1 respective to various pH at 70°C is matching emission calibration curve against pH at normal temperature. In vivo emission of sfGFPp1 shows better ratiometric characteristics compared to original superfolder GFP. Thus, T203C mutation along with superfolder mutations has unlocked GFP to become a successful probe in reporting intracellular pH of thermophiles.