Expression and purification of active mouse and human NEIL3 proteins.

Protein Expression and Purification
Minmin LiuSusan S Wallace

Abstract

Endonuclease VIII-like 3 (Neil3) is one of the five DNA glycosylases found in mammals that recognize and remove oxidized bases, and initiate the base excision repair (BER) pathway. Previous attempts to express and purify the mouse and human orthologs of Neil3 in their active form have not been successful. Here we report the construction of bicistronic expression vectors for expressing in Escherichia coli the full-length mouse Neil3 (MmuNeil3), its glycosylase domain (MmuNeil3Δ324), as well as the glycosylase domain of human Neil3 (NEIL3Δ324). The purified Neil3 proteins are all active, and NEIL3Δ324 exhibits similar glycosylase/lyase activity as MmuNeil3Δ324 on both single-stranded and double-stranded substrates containing thymine glycol (Tg), spiroiminodihydantoin (Sp) or an abasic site (AP). We show that N-terminal initiator methionine processing is critical for the activity of both mouse and human Neil3 proteins. Co-expressing an E. coli methionine aminopeptidase (EcoMap) Y168A variant with MmuNeil3, MmuNeil3Δ324 and NEIL3Δ324 improves the N-terminal methionine processing and increases the percentage of active Neil3 proteins in the preparation. The purified Neil3 proteins are suitable for biochemical, structural and function...Continue Reading

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Citations

Aug 2, 2013·Antioxidants & Redox Signaling·Timothy L ScottTadahide Izumi
Jan 1, 2013·Mutation Research·Minmin LiuSusan S Wallace
Oct 15, 2013·Environmental and Molecular Mutagenesis·Susan S Wallace
Mar 22, 2015·Mutation Research. Reviews in Mutation Research·Miral Dizdaroglu
Aug 13, 2013·Journal of the American Chemical Society·Paige L McKibbinSheila S David
Nov 2, 2016·The Journal of Clinical Investigation·Michel J MassaadRaif S Geha
Jan 18, 2017·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Lars Schomacher, Christof Niehrs
Sep 14, 2019·PloS One·Marcin RadomPiotr Formanowicz
May 5, 2020·Tumour Biology : the Journal of the International Society for Oncodevelopmental Biology and Medicine·Oanh Tn TranDawit Kidane
Mar 8, 2019·Nature·R Alex WuJohannes C Walter
Sep 4, 2020·The Journal of Biological Chemistry·Alyssa A RodriguezBrandt F Eichman
Apr 1, 2021·International Journal of Radiation Biology·Susan S Wallace
Jul 1, 2021·International Journal of Radiation Biology·Susan S Wallace
Jun 6, 2019·Neuro-degenerative Diseases·Petra SmatlikovaZdenka Ellederová

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