食品科学 ›› 2019, Vol. 40 ›› Issue (4): 138-145.doi: 10.7506/spkx1002-6630-20180128-385

• 生物工程 • 上一篇    下一篇

金黄色葡萄球菌新型肠毒素SEK原核表达、纯化及溶液构象分析

田万帆,刘?骥,赵燕英,唐俊妮*   

  1. (西南民族大学生命科学与技术学院,四川?成都 610041)
  • 出版日期:2019-02-25 发布日期:2019-03-05
  • 基金资助:
    国家自然科学基金面上项目(31371781);西南民族大学中央高校基本科研业务费重点项目(2018NZD14)

Prokaryotic Expression, Purification, and Solution Conformation of Staphylococcus aureus Enterotoxin K

TIAN Wanfan, LIU Ji, ZHAO Yanying, TANG Junni*   

  1. (College of Life Science and Technology, Southwest Minzu University, Chengdu 610041, China)
  • Online:2019-02-25 Published:2019-03-05

摘要: 金黄色葡萄球菌K型肠毒素(Staphylococcus aureus enterotoxin K,SEK)由金黄色葡萄球菌肠毒素基因簇编码,流行病学显示,编码SEK蛋白的sek基因在食品源金黄色葡萄球菌菌株中具有较高的检出率,说明SEK也可能是一种重要的食品中毒潜在致病因子。本研究将截去N端信号肽的SEK蛋白编码基因与原核表达载体pET-28a(+)连接,构建重组表达质粒pET-28a(+)-ΔNspsek;通过对sek基因在不同原核表达宿主菌中的表达及表达条件优化分析,确立SEK蛋白的最优表达条件;利用镍亲合层析纯化含组氨酸(His)标签的SEK融合蛋白,将凝血酶切除His标签后的SEK蛋白进行聚合状态及热稳定分析、荧光发射谱和圆二色谱分析。结果表明,重组蛋白SEK表达成功,热处理导致蛋白部分降解;荧光光谱揭示SEK蛋白在278?nm和295?nm波长处具有相同的最大色氨酸发射峰;344?nm波长处表明SEK蛋白处于紧密折叠的天然状态;圆二色谱分析表明,ΔNspSEK重组蛋白富含β-折叠(23.6%)、β-转角(28%)以及α-螺旋(29.1%)等二级结构。为深入研究SEK蛋白的结构与功能提供基础,也对改进食品加工工艺和提高食品安全具有指导意义。

关键词: 金黄色葡萄球菌K型肠毒素, 原核表达, 纯化, 热稳定性, 荧光发射谱, 圆二色谱

Abstract: Staphylococcus aureus enterotoxin K (SEK) is encoded by the enterotoxin gene cluster (egc) of S. aureus. Epidemiologic survey showed that the sek gene had a relatively high prevalence in foodborne isolates of S. aureus, which indicates that SEK protein may be an important virulence factor causing staphylococcal food poisoning. In this study, the sek gene from S. aureus without N-terminal signal peptide was firstly ligated into plasmid pET-28a(+). Then, the recombinant expression plasmid pET-28a(+)-ΔNspsek was transformed into competent cells of Escherichia coli. The expression conditions were optimized and the His-ΔNspSEK fusion protein was purified to homogeneity by Ni-Sepharose affinity chromatography. The purified protein without His tag was used to assay polymerization status, thermal stability, fluorescence emission and circular dichroism spectra. The results showed that the recombinant protein ΔNspSEK was successfully expressed and purified. It could be partially degraded by heat treatment. Fluorescence emission spectra of ΔNspSEK exhibited identical tryptophan emission peaks at 278 and 295 nm, as well as excitation at 344 nm indicating that ΔNspSEK is tightly folded in nature. Circular dichroism spectra revealed that His-ΔNspSEK tagged with 6 × His sequence was rich in β-sheet (23.6%), β-turn (28%), and α-helix (29.1%). The results of this investigation will facilitate further study of the structure and function of SEK, and also provide a useful guideline to improve food processing technology and food safety.

Key words: Staphylococcus aureus enterotoxin K, prokaryotic expression, purification, thermal stability, fluorescence emission spectra, circular dichroism

中图分类号: