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

• Bioengineering • Previous Articles     Next Articles

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

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

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