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Structural Analysis of the Effect of a Dual-FLAG Tag on Transthyretin.
Biochemistry ( IF 2.9 ) Pub Date : 2020-03-02 , DOI: 10.1021/acs.biochem.0c00105
Mehdi Shirzadeh 1 , Michael L Poltash 1 , Arthur Laganowsky 1 , David H Russell 1
Affiliation  

Recombinant proteins have increased our knowledge regarding the physiological role of proteins; however, affinity purification tags are often not cleaved prior to analysis, and their effects on protein structure, stability and assembly are often overlooked. In this study, the stabilizing effects of an N-terminus dual-FLAG (FT2) tag fusion to transthyretin (TTR), a construct used in previous studies, are investigated using native ion mobility-mass spectrometry (IM-MS). A combination of collision-induced unfolding and variable-temperature electrospray ionization is used to compare gas- and solution-phase stabilities of FT2-TTR to wild-type and C-terminal tagged TTR. Despite an increased stability of both gas- and solution-phase FT2-TTR, thermal degradation of FT2-TTR was observed at elevated temperatures, viz., backbone cleavage occurring between Lys9 and Cys10. This cleavage reaction is consistent with previously reported metalloprotease activity of TTR [Liz et al. 2009] and is suppressed by either metal chelation or excess zinc. This study brings to the fore the effect of affinity tag stabilization of TTR and emphasizes unprecedented detail afforded by native IM-MS to assess structural discrepancies of recombinant proteins from their wild-type counterparts.

中文翻译:

双FLAG标签对运甲状腺素蛋白影响的结构分析。

重组蛋白质增加了我们对蛋白质生理作用的认识。然而,亲和纯化标签通常在分析前不会被切割,并且它们对蛋白质结构,稳定性和组装的影响常常被忽略。在这项研究中,使用天然离子淌度质谱(IM-MS)研究了N端双FLAG(FT2)标签融合蛋白对运甲状腺素蛋白(TTR)(先前研究中使用的构建体)的稳定作用。碰撞诱导的展开和可变温度电喷雾电离的组合用于比较FT2-TTR与野生型和C末端标记TTR的气相和溶液相稳定性。尽管气相FT2-TTR和溶液相FT2-TTR的稳定性均得到提高,但在高温下仍观察到FT2-TTR的热降解,即 骨架裂解发生在Lys9和Cys10之间。该切割反应与先前报道的TTR的金属蛋白酶活性一致[Liz等人。2009],并且由于金属螯合或过量锌而受到抑制。这项研究突出了TTR亲和标签稳定化的作用,并强调了天然IM-MS提供的前所未有的细节来评估重组蛋白与野生型对应物之间的结构差异。
更新日期:2020-03-03
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