ISSN: 2641-9459
Authors: Jennifer Bernal, Zainab Ojutiku, Adriana Cordero, AshLee Martinez, Kayle Berkey and Kevin Francis*
Tetracyclines are an important group of antibiotics used in agricultural and clinical settings. They function by binding to the 30S subunit of the prokaryotic ribosome to effectively halt translation. The emergence of tetracycline resistant strains of pathogenic bacteria has threatened the continued use of the drug. 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 amounts of pure protein that can be obtained from current protocols (~6 mg/L of cell culture). The current report describes the development of optimized expression and purification protocols to obtain larger amounts of pure, soluble enzyme suitable for detailed kinetic studies to deduce its chemical mechanism. During the initial phase of this work the pET22a+ plasmid with the gene encoding for TetX was used to transform either DH5α or Rosetta (DE3) pLysS Escherichia coli cells. Expression trials of these cell lines in which both the temperature and times of incubation with isopropyl β-D-1-thiogalactopyrandoside were then carried out. Of the conditions tested, optimal expression of TetX was found with a 20 h induction period at 30 °C using E. coli DH5α cells. Ammonium sulfate precipitation trials were then conducted where it was concluded that a 20 fold purification of the enzyme is achieved through treatment with 40% saturation of salt and collection of the supernatant after centrifugation. A nickel affinity chromatographic protocol was also developed, which purified the enzyme to high levels as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the specific activity of the purified sample. The expression and purification protocol developed here resulted in ~36 mg/L of cell culture, which is a 6-fold improvement over published protocols.
Keywords: Protein Biochemistry; Enzyme Expression And Purification; Antibiotic Resistance; Enzyme Kinetic Assays
