بيان و خالص‌سازي آنزيم ترمينال دئوكسي نوكلئوتيديل ترانسفراز (TdT) نوتركيب و ارزيابي اثر گلايسين بر فعاليت آن

Expression and purification of recombinant deoxynucleotidyl transferase (TdT) enzyme and eva‎luation of the effect of glycine on its activity

زيست شناسي سلولي مولكولي و ميكروبيولوژي

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase belonging to the pol X polymerase family. Unlike other DNA polymerases in the nucleotidyl transferase family, this enzyme can catalyze the addition of natural and unnatural nucleotides to the 3ʹ hydroxyl end of single-stranded, double-stranded DNA, or RNA without a template strand. This unique advantage is widely utilized in the construction of biosensors. Like all polymerases, TdT requires the presence of a metal ion to catalyze the phosphorolysis reaction and can utilize various metal ions such as Co2+, Mn2+, Zn2+, and Mg2+. Due to the extensive applications of terminal deoxynucleotidyl transferase, including its use in TUNEL and immunofluorescence methods, the construction of biosensors and polynucleotides, the development of aptamers, and DNA nanostructures, studying this enzyme is of great importance. In this research, the effect of the stabilizing osmolyte glycine on the activity of TdT was investigated. To this end, after expressing and purifying the enzyme and confirming the expression accuracy using SDS-PAGE, the enzyme activity was eva‎luated by extending the length of a single-stranded sequence and constructing a random polynucleotide strand in the presence of various concentrations of glycine and at different pH levels. The results of this study showed that purified TdT enzyme has acceptable activity at pH 5 to 8. Also, although glycine had no significant effect on enzyme activity in the concentration range of 0.025 M to 0.1 M, it significantly reduced enzyme activity at concentrations higher than 0.1 M.

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