Development and application of gel electrophoresis methods for assaying protein kinetic stability

Thibeault, Jane
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Colón, Wilfredo
Barquera, Blanca L.
Royer, Catherine Ann
Koffas, Mattheos A. G.
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Biochemistry and biophysics
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The gel electrophoresis method D2D SDS-PAGE further exploits SDS-resistance and is coupled with matrix assisted laser desorption/ionization – time of flight mass spectrometry (MALDI-TOF MS) to explore the identity of KSPs in biological lysates. D2D SDS-PAGE was applied to cytoplasmic lysates from the bacterial pathogen model systems Pseudomonas aeruginosa and Staphylococcus aureus. While many of the identified KSPs possess roles commonly associated with kinetic stability, including stress response and primary metabolic functions, additional KSPs identified from P. aeruginosa are involved in toxin production and in conveying antibiotic resistance. A separate study explored the KSP profiles of the Bacteroides species, B. fragilis and B. thetaiotaomicron, common nanoaerobic members of the human gut microbiota. Through D2D SDS-PAGE visualization and MALDI-TOF MS, these bacteria were found to express the KSP, superoxide dismutase, at extremely high concentrations and contained several other KSPs involved in maintaining cellular redox homeostasis. It appears that these nanoaerobic bacteria are constantly primed to combat oxidative stress to protect themselves from damage and possibly to shield less aerotolerant organisms in the microbiota.
May 2019
School of Science
Biochemistry and Biophysics Program
Rensselaer Polytechnic Institute, Troy, NY
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