Messing Simon, Barnhart Kirsten, Drew Matthew, Granato-Guerrero Natalie, Grose Carissa, Higgins Brianna, Hong Min, Hull Jenna, Perkins Shelley, Poon Ivy, Ramakrishnan Nitya, Seabolt Amanda, Taylor Troy, Wall Vanessa E, Wright Nicholas, Gillette William, Esposito Dominic
Protein Expression Laboratory, NCI RAS Initiative, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.
Protein Expression Laboratory, NCI RAS Initiative, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.
Protein Expr Purif. 2025 Apr;228:106648. doi: 10.1016/j.pep.2024.106648. Epub 2024 Dec 15.
Tobacco-etch-virus (TEV) protease is the workhorse of many laboratories in which protein expression is the linchpin of downstream experiments. TEV protease is remarkable in its sequence specificity as the cleavage sequence rarely appears in higher organisms and its ability to cleave fusion tag proteins from proteins of interest. Herein we report work done on large-scale production of TEV protease using different promotors, media, fusion tags, and expression platforms. During our work we detected post-translational modification (gluconoylation and phosphogluconoylation) of TEV protease and the subsequent effects this has on the purity of the protein. Subsequently we made our pgl plus bacteria that negates these modifications and their effects. We also introduce a GFP-based assay for measurement of activity and ultimately a new set of protocols for producing 400-500 mg/L TEV protease.
烟草蚀纹病毒（TEV）蛋白酶是许多实验室的得力工具，在这些实验室中，蛋白质表达是下游实验的关键。TEV蛋白酶在序列特异性方面表现出色，因为其切割序列在高等生物中很少出现，并且它能够从目标蛋白质上切割融合标签蛋白。在此，我们报告了使用不同启动子、培养基、融合标签和表达平台大规模生产TEV蛋白酶的工作。在我们的工作过程中，我们检测到了TEV蛋白酶的翻译后修饰（葡糖酰化和磷酸葡糖酰化）以及这对蛋白质纯度的后续影响。随后，我们构建了能够消除这些修饰及其影响的pgl plus细菌。我们还引入了一种基于绿色荧光蛋白（GFP）的活性测定方法，并最终制定了一套新的方案，用于生产浓度为400 - 500 mg/L的TEV蛋白酶。
