کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020600 1069193 2012 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Expression and purification of active mouse and human NEIL3 proteins
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Expression and purification of active mouse and human NEIL3 proteins
چکیده انگلیسی

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.


► We expressed and purified active mouse and human Neil3 proteins with high purity.
► Co-expression of EcoMapY168A improves N-terminal methionine processing.
► Co-expression of EcoMapY168A increases the percentage of active Neil3 proteins.
► NEIL3Δ324 exhibits similar glycosylase/lyase activity to MmuNeil3Δ324.
► This protocol is valuable for future structural and functional studies of Neil3.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 84, Issue 1, July 2012, Pages 130–139
نویسندگان
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