Liu Minmin, Bandaru Viswanath, Holmes Alicia, Averill April M, Cannan Wendy, Wallace Susan S
Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Dr. Burlington, VT 05405-0086, USA.
Protein Expr Purif. 2012 Jul;84(1):130-9. doi: 10.1016/j.pep.2012.04.022. Epub 2012 May 5.
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 functional studies.
内切核酸酶VIII样3（Neil3）是在哺乳动物中发现的五种DNA糖基化酶之一，可识别并去除氧化碱基，并启动碱基切除修复（BER）途径。此前尝试以活性形式表达和纯化Neil3的小鼠和人类直系同源物均未成功。在此，我们报告了双顺反子表达载体的构建，用于在大肠杆菌中表达全长小鼠Neil3（MmuNeil3）、其糖基化酶结构域（MmuNeil3Δ324）以及人类Neil3的糖基化酶结构域（NEIL3Δ324）。纯化后的Neil3蛋白均具有活性，并且NEIL3Δ324在含有胸腺嘧啶乙二醇（Tg）、螺环亚氨基二氢尿嘧啶（Sp）或无碱基位点（AP）的单链和双链底物上表现出与MmuNeil3Δ324相似的糖基化酶/裂解酶活性。我们表明，N端起始甲硫氨酸的加工对于小鼠和人类Neil3蛋白的活性至关重要。与MmuNeil3、MmuNeil3Δ324和NEIL3Δ324共表达大肠杆菌甲硫氨酸氨基肽酶（EcoMap）Y168A变体可改善N端甲硫氨酸的加工，并增加制备物中活性Neil3蛋白的比例。纯化后的Neil3蛋白适用于生化、结构和功能研究。
