Development of a BLI assay-based method for detecting LptA/LptC interaction.

Xiaowei Dai; Xiaohong Zhu; Shuyi SI; Yan LI; Lijie Yuan

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Development of a BLI assay-based method for detecting LptA/LptC interaction.

Author: Xiaowei DAI ¹ ; Xiaohong ZHU ² ; Shuyi SI ² ; Yan LI ² ; Lijie YUAN ¹
Author Information

1. Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Disease, Hebei Key Laboratory for Chronic Diseases, School of Basic Medical Science, North China University of Science and Technology, Tangshan 063210, Hebei, China.
2. National Center for Drug (Microbiology) Screening Laboratory, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Publication Type:Journal Article
Keywords: LptA; LptA/LptC interaction; LptC; biolayer interferometry (BLI); lipopolysaccharide (LPS); lipopolysaccharide transport (Lpt)
MeSH: Carrier Proteins; Escherichia coli/metabolism*; Escherichia coli Proteins/metabolism*; Interferometry; Membrane Proteins/metabolism*
From: Chinese Journal of Biotechnology 2021;37(9):3300-3309
CountryChina
Language:Chinese
Abstract: In Gram-negative bacteria, lipopolysaccharide transport (Lpt) protein LptA and LptC form a complex to transport LPS from the inner membrane (IM) to the outer membrane (OM). Blocking the interaction between LptA and LptC will lead to the defect of OM and cell death. Therefore, Lpt protein interaction could be used as a target to screen new drugs for killing Gram-negative bacteria. Here we used biolayer interferometry (BLI) assay to detect the interaction between LptA and LptC, with the aim to develop a method for screening the LptA/LptC interaction blockers in vitro. Firstly, LptC and LptA with or without signal peptide (LptAfull or LptAno signal) were expressed in E. coli BL21(DE3). The purified proteins were then labeled with biotin and the super streptavidin (SSA) biosensor was blocked with diluent. The biotin labeled protein sample was mixed with the sensor, and then the binding of the protein with a series of diluted non biotinylated protein was detected. At the same time, non-biotinylated protein was used as a control. The binding of biotinylated protein to a small molecule IMB-881 and the blocking of interaction were also detected by the same method. In the blank control, the biosensor without biotinylated protein was used to detect the serially diluted samples. The signal response constant was calculated by using steady analysis. The results showed that biotinylated LptC had a good binding activity with LptAfull and LptAno signal with KD value 2.9e⁻⁷±7.9e⁻⁸ and 6.0e⁻⁷±2.8e⁻⁸, respectively; biotinylated LptAno signal had a good binding activity with LptC, with a KD value of 9.6e⁻⁷±7.2e⁻⁸. All binding curves showed obvious fast binding and fast dissociation morphology. The small molecule compound IMB-881 can bind to LptA to block the interaction between LptA and LptC, but has no binding activity with LptC. In summary, we developed a method for detecting the LptA/LptC interaction based on the BLI technology, and confirmed that this method can be used to evaluate the blocking activity of small molecule blockers, providing a new approach for the screening of LptA/LptC interaction blockers.