Nikoomanzar Ali, Dunn Matthew R, Chaput John C
Department of Pharmaceutical Sciences. University of California, Irvine, California.
Curr Protoc Nucleic Acid Chem. 2017 Jun 19;69:4.75.1-4.75.20. doi: 10.1002/cpnc.33.
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.
聚合酶工程使得合成具有多样主链结构和化学功能的异源核酸聚合物（XNA）成为可能。在DNA和XNA之间来回复制遗传信息的能力催生了一个名为合成遗传学的新科学领域，该领域旨在研究人工遗传聚合物中的遗传和进化的遗传概念。由于合成XNA聚合物所需的许多聚合酶无法从商业渠道获得，研究人员必须将这些酶作为重组蛋白从大肠杆菌中表达并纯化出来。本单元详细介绍了表达、纯化和评估具有改变的底物识别特性的工程聚合酶活性所需的步骤。该方案需要6天完成，每升大肠杆菌细菌培养物可产生约20毫克纯的、无核酸酶的聚合酶。© 2017约翰威立父子公司。
