大肠杆菌
突变体
细菌
噬菌体
半胱氨酸
化学
沙门氏菌
肠沙门氏菌
重组DNA
基质(水族馆)
生物化学
生物
酶
基因
生态学
遗传学
作者
Amit Singh,Sunil K. Arya,Nick Glass,Pejman Hanifi-Moghaddam,Ravendra Naidoo,Christine M. Szymanski,Jamshid Tanha,Stéphane Evoy
标识
DOI:10.1016/j.bios.2010.05.024
摘要
We report the use of genetically engineered tailspike proteins (TSPs) from the P22 bacteriophage for the sensitive and selective detection of Salmonella enterica serovar Typhimurium. High yields of two mutant TSPs, one with an N-terminal cysteine (N-Cys) and another with a C-terminal cysteine (C-Cys), have been obtained using recombinant protein expression and purification in Escherichia coli. The mutant TSPs did not have the native endorhamnosidase enzymatic activity of intact P22 phage as well as wild type TSPs (wtTSPs). We have used the Cys-tag to immobilize these TSPs onto gold coated surfaces using thiol-chemistry. Our results demonstrate that the N-Cys configuration of TSPs gives a bacterial capture density of 25.87 ± 0.61 bacteria/100 μm2 while the C-Cys configuration shows a density of 8.57 ± 0.19 bacteria/100 μm2. This confirms that the appropriate orientation of the TSPs on the surface is important for efficient capture of the host bacteria. The bacterial capture density of the mutant N-Cys TSP was also 6-fold better than that obtained for intact P22 phage as well as wtTSPs. Bovine-serum albumin was used as a protective layer to prevent any non-specific binding of the bacteria onto the gold substrate. The recognition specificity was confirmed using 3 strains of E. coli which showed negligible binding. In addition, the host bacteria did not show any binding in the absence of the TSPs on the surface. We further show a selective real-time analytical detection of Salmonella by N-Cys mTSP-immobilized on gold coated SF-10 glass plates using surface plasmon resonance. The sensitivity of detection was found to be 103 cfu/ml of bacteria.
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