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Development of a glutathione-S-transferase-based biosensor for the detection of heavy metals

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2005
Saatçi, Ebru
In the recent years, environmental pollution becomes a health threatening issue for human beings. Technological developments introduce industrial wastes and heavy metals, and developments in agriculture introduce pesticides into the world that we live. All these toxic wastes accumulate in drinking water and food consumed by humans. Therefore, detection of toxic wastes in all kinds of environmental samples, and development of new detection techniques become an important issue. In this study, development of a protein-based biosensor for detection of heavy metals in environmental samples, by expressing genetically modified glutathione S-transferase (GST-(His)6) protein in E.Coli BL21 (DE3) expression system, was designed. Recombinant GST proteins was expressed in E.Coli BL21 (DE3) expression system and purified with Glutathione Sepharose 4B affinity column and Ni-NTA spin kit. GST activities were determined using the GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). Protein expression was tested by SDS-PAGE and Western blot analysis. Product formation linearly increased up to 1 mM CDNB, 1 mM GSH, 1.7 æg proteins in 0.05 M, pH 6.9 phosphate buffer in the final volume of 1.0 ml at 25?C. The Vmax and Km values for GST-(His)6 towards CDNB and GSH were calculated with Lineweaver-Burk as CDNB Vmax; 22.88 æmol/min/mg, Km; 4.29 mM,and as GSH Vmax; 6.42 æmol/min/mg, 24.45 æmol/min/mg, Km; 3.69 mM, respectively. Biosensor working electrode was prepared by immobilizing the GST-(His)6 by 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) coupling on the gold surface. Electrode preparation was confirmed by cyclic voltammetry measurements. The biosensor was inserted as the working electrode in the constructed three(four)-electrode flow cell. The conformational change resulting from the binding of the metal ions to the recombinant protein causing a capacitance change