鼠李糖乳杆菌GG基础菌毛蛋白SpaE的结晶和X射线衍射分析。

PubMed ID
发表日期 2017年Jun月

原始出处 晶体学报。F节,结构生物学通信
Acta crystallographica. Section F, Structural biology communications
作者 Mishra  Arjun K  Megta  Abhin Kumar  Palva  Airi  von Ossowski  Ingemar  Krishnan  Vengadesan 

文献标题 鼠李糖乳杆菌GG基础菌毛蛋白SpaE的结晶和X射线衍射分析。
Crystallization and X-ray diffraction analysis of SpaE, a basal pilus protein from the gut-adapted Lactobacillus rhamnosus GG.

文献摘要

SpaE是来源于鼠李糖乳杆菌(Lactobacillus rhamnosus GG)肠道的、依赖于蔗糖酶的spaed菌毛中预测的基础菌毛亚单位。到目前为止,细胞壁锚定的基生菌毛的结构特征仍然很难确定,而且仅限于病原属和种的少数例子。为了进一步从结构上了解索拉酶依赖性菌毛在有害细菌中的锚定和组装的分子机制,我们在大肠杆菌中通过重组表达制备了用于结晶的鼠李糖乳杆菌GG-SpaE。尽管几次结晶SpaE蛋白的尝试都没有成功,但最终以PEG 3350为沉淀剂和高蛋白浓度制备出了分辨率为3.1 的三角晶体。通过添加剂的进一步优化,产生了斜方晶形SpaE晶体,其衍射分辨率高达1.5 。为了加速SAD相的结构测定,生长了硒取代(正交)SpaE晶体,并收集了1.8  分辨率的X射线衍射数据。


SpaE is the predicted basal pilin subunit in the sortase-dependent SpaFED pilus from the gut-adapted and commensal Lactobacillus rhamnosus GG. Thus far, structural characterization of the cell-wall-anchoring basal pilins has remained difficult and has been limited to only a few examples from pathogenic genera and species. To gain a further structural understanding of the molecular mechanisms that are involved in the anchoring and assembly of sortase-dependent pili in less harmful bacteria, L. rhamnosus GG SpaE for crystallization was produced by recombinant expression in Escherichia coli. Although several attempts to crystallize the SpaE protein were unsuccessful, trigonal crystals that diffracted to a resolution of 3.1 Å were eventually produced using PEG 3350 as a precipitant and high protein concentrations. Further optimization with a combination of additives led to the generation of SpaE crystals in an orthorhombic form that diffracted to a higher resolution of 1.5 Å. To expedite structure determination by SAD phasing, selenium-substituted (orthorhombic) SpaE crystals were grown and X-ray diffraction data were collected to 1.8 Å resolution.


获取全文 10.1107/S2053230X17006963