Overexpressing and Purifying a Toxic Nuclease from Escherichia coli

Our lab aims to structurally and biochemically characterize a conserved endonuclease found in nidoviruses, including coronaviruses, to develop an evolutionary model and provide a basis for therapeutic targets. Nucleases can be difficult to express in E coli systems due to enzymatic activity on cellular DNA or RNA, which can result in slow growth and poor protein yields. Our finding will allow us to purify toxic nucleases from other nidoviruses for further downstream biochemical and structural studies.

To begin, use one 50 milliliter sterilized Erlenmeyer flask for each liter of culture and prepare two to three additional flasks as starter cultures. Label one of the flasks with a star to designate it for optical density checks, as its measurements will represent the entire growth, unless there is a visual difference among flasks. Using a graduated cylinder, prepare a master mix by combining 60 milliliters of 2X TY medium and 60 microliters of ampicillin stock solution thawed from minus 20 degrees Celsius.