Cautionary note on ribonuclease activity of recombinant PR-10 proteins
Cautionary note on ribonuclease activity of recombinant PR-10 proteins
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DOI：
10.1101/2023.02.27.529914
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发表时间：
2023-02
期刊：
影响因子：
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通讯作者：
Rawit Longsaward;N. Sanguankiattichai;Unchera Viboonjun;R. V. D. van der Hoorn
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作者：
Rawit Longsaward;N. Sanguankiattichai;Unchera Viboonjun;R. V. D. van der Hoorn
我们研究了来自橡胶树（Hevea brasiliensis）的PR-10的三种剪接异构体的生化性质，发现纯化的重组HbPR 10可以在体外引起RNA降解，这是许多PR-10蛋白所描述的众所周知的活性。对于所有三种HbPR 10剪接亚型都观察到了这种核糖核酸酶活性，并且通过煮沸而被消除。然而，包括阴性对照蛋白质揭示核糖核酸酶活性更确切地来源于从大肠杆菌共纯化的RNA酶。大肠杆菌中的RNA酶抑制剂、热灭活蛋白、阴性对照蛋白等传统方法所忽略的蛋白质。在全世界不同实验室发表的关于PR-10蛋白中核糖核酸酶活性的至少9份报告中，缺少关键的对照蛋白，这表明在核糖核酸酶测定中经常忽略适当的对照。所提出的警示适用于几种具有宣称的核糖核酸酶活性的PR-10蛋白，并要求使用不同的测定和突变PR-10蛋白作为对照。
We studied the biochemical properties of three splicing isoforms of PR-10 from rubber tree (Hevea brasiliensis) and found that purified recombinant HbPR10 can cause RNA degradation in vitro, a well-known activity described for many PR-10 proteins. This ribonuclease activity was observed for all three HbPR10 splicing isoforms and is abolished by boiling. However, inclusion of a negative control proteins revealed that ribonuclease activity rather originates from RNases that are copurified from E. coli, which are overlooked by traditionally used controls such as heat inactivation, RNase inhibitors and negative control proteins obtained with different procedures. The crucial control proteins are missing for at least nine reports on ribonuclease activity in PR-10 proteins published by different laboratories worldwide, indicating that proper controls are frequently overlooked in ribonuclease assays. The raised cautionary note applies to several PR-10 proteins with proclaimed ribonuclease activities and call for the use of different assays and mutant PR-10 proteins as control.