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Abstract：
Recombinant Francisella tularensis universal stress protein with a C-terminal histidine-tag (rUsp/His6) was expressed in Escherichia coli. Endogenous F. tularensis Usp has a predicted molecular mass of 30 kDa, but rUsp/His6 had an apparent molecular weight of 33 kDa based on Western blot analyses. To determine the source of the higher molecular weight for rUsp/His6, post translational modifications were examined. Tryptic peptides of purified rUsp/His6 were subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) and fragmentation spectra were searched for acetylated lysines and polyaminated glutamines. Of the 24 lysines in rUsp/His6, 10 were acetylated (K63, K68, K72, K129, K175, K201, K208, K212, K233, and K238) and three of the four glutamines had putrescine, spermidine and spermine adducts (Q55, Q60 and Q267). The level of post-translational modification was substoichiometric, eliminating the possibility that these modifications were the sole contributor to the 3 kDa extra mass of rUsp/His6. LC-MS/MS revealed that stop codon readthrough had occurred resulting in the unexpected addition of 20 extra amino acids at the C-terminus of rUsp/His6, after the histidine tag. Further, the finding of polyaminated glutamines in rUsp/His6 indicated that E. coli is capable of transglutaminase activity.
摘要：
在大肠杆菌中表达了具有C末端组氨酸标签的重组土拉弗朗西斯菌通用应激蛋白（rUsp/His6）。内源F.tularensisUsp的预测分子量为30kDa，但根据Western印迹分析，rUsp/His6的表观分子量为33kDa。为了确定rUsp/His6的较高分子量的来源,检查翻译后修饰。将纯化的rUsp/His6的胰蛋白酶肽进行液相色谱串联质谱（LC-MS/MS），并搜索乙酰化赖氨酸和多聚胺化谷氨酰胺的片段谱。在rUsp/His6中的24个赖氨酸中，有10个被乙酰化（K63，K68，K72，K129，K175，K201，K208，K212，K233和K238），并且四种谷氨酰胺中的三种具有腐胺，亚精胺和精胺加合物（Q55，Q60和Q267）。翻译后修饰的水平是亚化学计量的，消除了这些修饰是rUsp/His6的3kDa额外质量的唯一原因的可能性。LC-MS/MS揭示终止密码子连读已经发生,导致在组氨酸标签之后,在rUsp/His6的C-末端意外地添加了20个额外的氨基酸。Further,rUsp/His6中的多胺化谷氨酰胺的发现表明大肠杆菌能够具有转谷氨酰胺酶活性。
