Engineered Polymerases with Altered Substrate Specificity: Expression and Purification

Polymerase engineering is making it possible to synthesize xeno-nucleic acid polymers (XNAs) with diverse backbone structures and chemical functionality. The ability to copy genetic information back and forth between DNA and XNA has led to a new field of science known as synthetic genetics, which aims to study the genetic concepts of heredity and evolution in artificial genetic polymers. Since many of the polymerases needed to synthesize XNA polymers are not available commercially, researchers must express and purify these enzymes as recombinant proteins from E. coli. This unit details the steps needed to express, purify, and evaluate the activity of engineered polymerases with altered substrate recognition properties. The protocol requires 6 days to complete and will produce ∼20 mg of pure, nuclease-free polymerase per liter of E. coli bacterial culture. © 2017 by John Wiley & Sons, Inc.