嗜酸嗜热古菌Sulfolobus acidocaldarius编码尿嘧啶DNA糖苷酶表达，纯化与酶学特征

Cloning，expression，purification and characterization of two uracil-DNA glycosylases from Sulfolobus acidocaldarius

摘要:

摘要:【目的】嗜高温微生物面临dC脱氨基生成dU损伤的巨大压力，鉴定嗜酸嗜热古菌S.acidocaldarius来源的尿嘧啶DNA糖苷酶(UDG)切除dU损伤的酶学活性。【方法】重组表达来源于S.acidocaldarius的IV和V型UDG，经亲和纯化得到电泳纯重组蛋白。然后利用人工合成的dU(deoxyuracil)修饰寡核苷酸片段作为底物，体外鉴定两种重组UDG 的酶学特性。【结果】来源于S.acidocaldarius的IV和V型重组UDG具有相似的酶学特性。IV型UDG催化效率更高，比活性是V型重组UDG的750倍左右。作为来自嗜热微生物 的蛋白，S.acidocaldarius的IV和V型UDG的最适反应温度为65－75℃。【结论】IV型UDG比V型UDG水解dU碱基和脱氧核糖之间糖苷键的能力更强。

Abstract:

Abstract: ［Objective］ To characterize uracil-DNA glycosylase from acidophilic and thermophilic Sulfolobus acidocaldarius.［Methods］We cloned udgIV and udgV genes from S.acidocaldarius，expressed the two recombinant UDG proteins in E.coli species BL21 (DE3) Rosetta-pLysS，purified the recombinant UDGs and characterized the removal of dU by UDGs.［Results］We successfully expressed two S.acidocaldarius UDGs and found both UDGs having the activity of dU removal. In comparison to UDGV，UDGIV was more efficient in dU removal，with a 750-foldactivity.［Conclusion］In comparison to UDGV，UDGIV from S． acidocaldarius was a more efficient enzyme responsible for the removal of dU from DNA in vitro.