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Recombinant protein production and purification of SiiD, SiiE and SiiF - Components of the SPI4-encoded type I secretion system from Salmonella Typhimurium.
Protein Expression and Purification ( IF 1.6 ) Pub Date : 2020-04-03 , DOI: 10.1016/j.pep.2020.105632 Stefan Klingl 1 , Sina Kordes 1 , Benedikt Schmid 1 , Roman G Gerlach 2 , Michael Hensel 3 , Yves A Muller 1
Protein Expression and Purification ( IF 1.6 ) Pub Date : 2020-04-03 , DOI: 10.1016/j.pep.2020.105632 Stefan Klingl 1 , Sina Kordes 1 , Benedikt Schmid 1 , Roman G Gerlach 2 , Michael Hensel 3 , Yves A Muller 1
Affiliation
In humans, Salmonella enterica infections are responsible for a plethora of medical conditions. These include intestinal inflammation and typhoid fever. The initial contact between Salmonella and polarized epithelial cells is established by the SPI4-encoded type I secretion system (T1SS), which secretes SiiE, a giant non-fimbrial adhesin. We have recombinantly produced various domains of this T1SS from Salmonella enterica serovar Typhimurium in Escherichia coli for further experimental characterization. We purified three variants of SiiD, the periplasmic adapter protein spanning the space between the inner and outer membrane, two variants of the SiiE N-terminal region and the N-terminal domain of the SiiF ATP-binding cassette (ABC) transporter. In all three proteins, at least one variant yielded high amounts of pure soluble protein. Secondary structure content and cooperative unfolding were investigated by circular dichroism (CD) spectroscopy. Secondary structure contents were in good agreement with estimates derived from SiiD and SiiF homology models or, in case of the SiiE N-terminal region, a secondary structure prediction. For one SiiD variant, protein crystals could be obtained that diffracted X-rays to approximately 4 Å resolution.
中文翻译:
重组蛋白的生产和SiiD,SiiE和SiiF的纯化-鼠伤寒沙门氏菌SPI4编码的I型分泌系统的组成部分。
在人类中,肠沙门氏菌感染是造成许多医疗状况的原因。这些包括肠道炎症和伤寒。沙门氏菌和极化的上皮细胞之间的初始接触是通过SPI4编码的I型分泌系统(T1SS)建立的,该系统分泌一种巨大的非纤维粘附素SiiE。我们已经在大肠杆菌中从肠炎沙门氏菌血清鼠伤寒沙门氏菌重组产生了该T1SS的多个结构域,用于进一步的实验表征。我们纯化了SiiD的三个变体,即跨越内膜和外膜之间空间的周质衔接蛋白,SiiE N末端区域的两个变体和SiiF ATP结合盒(ABC)转运蛋白的N末端结构域。在所有三种蛋白质中,至少一种变体产生大量的纯可溶蛋白质。通过圆二色性(CD)光谱研究二级结构含量和协同展开。二级结构的内容与从SiiD和SiiF同源性模型得出的估计值非常吻合,或者在SiiE N端区域的情况下,二级结构预测也是如此。对于一个SiiD变体,可以获得将X射线衍射到大约4Å分辨率的蛋白质晶体。
更新日期:2020-04-03
中文翻译:
重组蛋白的生产和SiiD,SiiE和SiiF的纯化-鼠伤寒沙门氏菌SPI4编码的I型分泌系统的组成部分。
在人类中,肠沙门氏菌感染是造成许多医疗状况的原因。这些包括肠道炎症和伤寒。沙门氏菌和极化的上皮细胞之间的初始接触是通过SPI4编码的I型分泌系统(T1SS)建立的,该系统分泌一种巨大的非纤维粘附素SiiE。我们已经在大肠杆菌中从肠炎沙门氏菌血清鼠伤寒沙门氏菌重组产生了该T1SS的多个结构域,用于进一步的实验表征。我们纯化了SiiD的三个变体,即跨越内膜和外膜之间空间的周质衔接蛋白,SiiE N末端区域的两个变体和SiiF ATP结合盒(ABC)转运蛋白的N末端结构域。在所有三种蛋白质中,至少一种变体产生大量的纯可溶蛋白质。通过圆二色性(CD)光谱研究二级结构含量和协同展开。二级结构的内容与从SiiD和SiiF同源性模型得出的估计值非常吻合,或者在SiiE N端区域的情况下,二级结构预测也是如此。对于一个SiiD变体,可以获得将X射线衍射到大约4Å分辨率的蛋白质晶体。