Purification of Tetracycline Monooxygenase (TetX)
Abstract: Tetracycline is an antibiotic that treats infectious diseases triggered by pathogenic bacteria that range from acne to bacterial pneumonia. It functions by binding to the 30S subunit of the prokaryotic ribosome to effectively stop protein synthesis from occurring. The recent emergence of tetracycline resistant strains of pathogens has hampered its continued use in clinical settings. One mechanism of tetracycline resistance is the degradation of the drug by the flavin dependent enzyme tetracycline monooxygenase (TetX). Only a limited number of studies of the enzyme have been conducted to date due to the limited yields of pure protein that can be obtained from current protocols (~6 mg/L of cell culture). The objective of this research is to develop a purification protocol for the enzyme for mechanistic studies. The long-term goal of this research is to create an inhibitor of TetX for the purpose of finding a better treatment plan toward infectious diseases caused by tetracycline resistant strains of bacteria. With the recent development of an optimization protein expression protocol, a plasmid encoding TetX (pET22a – TetX) was successfully transformed into competent Escherichia coli cells with the selective use of the DH5α cell strain. Transformants were screened for optimal expression after induction for 20 h at 30 °C through measurements of the TetX dependent consummation of oxygen and quantification of the total protein in cell extracts. Permanent glycerol stock of a transformant that had a specific activity of 1,670 U/mg was prepared. Ammonium sulfate precipitation trials were then carried out where it was concluded that partial purification of the enzyme can be achieved through treatment with 40% salt and collection of the supernatant after centrifugation at 12,000 x g for 20 minutes. A nickel affinity chromatographic protocol was also developed which resulted in a purification of 2. The expression and purification protocol developed here resulted in 30 mg/L of cell culture, which is a 5-fold improvement over published protocols.
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