Journal of Biochemistry and Biotechnology mainly focuses on updating and advancing biological science through our high-quality articles which include original research, systematic reviews, meta-analyses, and technical reports which come under the scope of biochemistry and biotechnology.

Dr. Khue Vu Nguyen has written a research article about the Immobilization of firefly luciferase on the cell plasma membrane as a quantitative biosensor for measurement of ATP in the pericellular space in live mammalian cells. The purinergic signaling system consists of transporters, enzymes, and receptors responsible for the synthesis, release, action and extracellular inactivation of adenosine-5’-triphosphate (ATP) and its extracellular breakdown product adenosine. Up to date, the full appreciation of the role of ATP as an extracellular signal has been hampered by a lack of proper biosensors for accurate local real-time measurement of extracellular ATP concentration in the pericellular space from individual cells under physiological and pathological conditions. We describe herein the development of simple, sensitive, and reliable dual-function biosensor for the local real-time measurement of extracellular ATP concentration in the pericellular space in live HEK 293 cells by performing an immobilization of firefly luciferase (Fluc) coupled with the green fluorescent protein (GFP) on the plasma membrane of HEK 293 cells via a glycosyl-phosphatidylinositol, GPI, anchor derived from the human folate receptor 1 (FOLR1) protein: <5 μM pmeLUC. Our pmeLUC2 dual-function reporter construct was shown to be fluorescent and bioluminescent and could detect pericellular ATP at concentrations under physiological conditions: and the apparent KM of immobilized Fluc for ATP is to be 2-3 × 10-6 M, values much lower than the 51 × 10-6 M found for the free Fluc. In addition, there was no loss of immobilized Fluc activity in pmeLUC2 after more than 15 ATP measurements followed by 90 days stored at +4°C in PBS. The method used for the construction of our pmeLUC2 probe may pave the way for new strategies applicable to rational pmeLUC design. Its use in live cells and organisms, especially for identifying a new pathway for ATP secretion as a signaling molecule, promise to further expand its utility.

The presence of the immobilized firefly luciferase (Fluc) on the cell plasma membrane of the pmeLUC2 transfected HEK293 cells was confirmed by (a) the presence of GFP expression seen only on the surface of the pmeLUC2 transfected HEK293 cells as many dots (clusters), compared with that seen throughout the positive control of free GFP: vector 9 transfected HEK 293 cells and (b) the detection and measurement of the luminescence emission from the pmeLUC2 transfected HEK293 “ghost” (dead cell in which the outline remains visible, but without the cytosol as well as other cytoplasmic structures or stainable nucleus obtained after the cell lysis). Our pmeLUC2 dual-function reporter construct appears as suited, sensitive, and reliable biosensors for the local real-time measurement of extracellular ATP concentration in the pericellular space in live mammalian cells. The method used for the construction of our pmeLUC2 probe may pave the way for new strategies applicable to rational pmeLUC design. Its use in live cells and organisms, especially for identifying a new pathway for ATP secretion as a signaling molecule, promise to further expand its utility.

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Journal of Biochemistry & Biotechnology
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