Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Binding of the His-tagged Tail Protein J of Bacteriophage Lambda with Escherichia coli K-12

히스티딘으로 표지된 람다 박테리오파아지 꼬리 단백질 J와 대장균 K-12와의 결합

  • Shin, Hae Ja (Division of Energy and Bio-engineering, Dongseo University)
  • 신혜자 (동서대학교 에너지생명공학부 에너지환경공학전공)
  • Received : 2017.10.09
  • Accepted : 2017.12.19
  • Published : 2018.01.30
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Abstract

Detection of pathogenic microorganisms takes several days by conventional methods. It is necessary to assess microorganisms in a timely manner to reduce the risk of spreading infection. For this purpose, bacteriophages are chosen for use as a biosensing tool due to their host specificity, wide abundance, and safety. However, their lytic cycle limits their efficacy as biosensors. Phage proteins involved in binding to bacteria could be a robust alternative in resolving this drawback. Here, a fragment of tail protein J (residues 784 to 1,132) of phage lambda fused with 6X His-tag (6HN-J) at its N-terminus was cloned, overexpressed, purified, and characterized for its binding with microorganisms. The purified protein demonstrated a size of about 38 kDa in sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and bound with anti-His monoclonal antibodies. It bound specifically to Escherichia coli K-12, and not Salmonella typhimurium, Bacillus subtilis, or Pseudomonas aeruginosa in dot blotting. Binding of the protein to E. coli K-12 inhibited about 50% of the in vivo adsorption of the phage lambda to host cells at a concentration of $1{\mu}g/ml$ 6HN-J protein and almost 100% at $25{\mu}g/ml$ 6HN-J. The results suggest that a fusion viral protein could be utilized as a biosensing element (e.g., protein chips) for detecting microorganisms in real time.

본 연구에서는 미생물 검출용 바이오센서 제작을 위해 재조합 람다 파아지 꼬리 단백질 J을 센싱 요소로 활용가능한지를 조사하였다. 융합 단백질의 입체 장애를 최소화하기 위해 J 단백질 절편의 N-말단을 6X His-tag로 융합하고 HisTALONTM 컬럼으로 정제하였다. 정제 단백질은 약 38 kDa 크기를 SDS-PAGE에서 나타내며 anti-His 단클론 항체와 반응하였다. Anti-His 단클론 항체는 6HN-J를 처리한 E. coli K-12와 특이적으로 결합하나 BSA 처리하거나 6HN-J 처리한 다른 미생물들(Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa)과는 결합되지 않음을 보여주었다. 또한 정제 단백질과 숙주세포의 결합은 온전한 람다 파아지의 in vivo 숙주 표면 흡착을 약 $1{\mu}g/ml$ 단백질 농도에서 50%, $25{\mu}g/ml$ 단백질 농도에서 거의 완전히 방해하였다. 결론적으로 재조합 6HN-J 단백질은 탁월한 선택성과 선별성으로 인해 바이오센서의 제작에서 센싱 요소로 활용 가능함을 시사한다.

Keywords

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