Construction of a fusion protein consisting of α-1,3-glucan-binding domains and tetrameric red fluorescent protein, which is involved in the aggregation of α-1,3-glucan and inhibition of fungal biofilm formation
Journal of Bioscience and Bioengineering(2022)
Abstract
Agl-KA, an alpha-1,3-glucan-hydrolyzing enzyme from Bacillus circulans KA-304, has three alpha-1,3-glucan-binding domains DS1, CB6, and DS2 (DCD). While their individual binding activities toward insoluble alpha-1,3-glucan and fungal cell-wall are weak, the three domains in combination bind strongly to the alpha-1,3-glucan and the cell-wall. In this study, we constructed DCD-tetraRFP by fusing DCD with DsRed-Express2, a tetrameric red fluorescent protein. DCD-tetraRFP forms a tetramer in an aqueous solution and contains twelve substrate-binding domains in one complex. We also constructed DCD-monoGFP by fusing DCD with AcGFP1, a monomeric green fluorescent protein. The molecular weight of DCD-tetraRFP and DCD-monoGFP were compared. The results of gel filtration chromatography and dynamic light scattering indicated that DCD-tetraRFP was larger than DCD-monoGFP, suggesting that DCD-tetraRFP had a tetrameric structure. In addition, DCD-tetraRFP bound to insoluble alpha-1,3-glucan strongly, and the amount of DCD-tetraRFP binding to 0.01% alpha-1,3-glucan was about twice of DCD-monoGFP. The K-d values of DCD-tetraRFP (measurements per subunit) and DCD-monoGFP were 0.16 and 0.84 mu M, respectively. Adding DCD-tetraRFP to a suspension of alpha-1,3-glucan caused glucan aggregation; however, adding DCD-monoGFP did not. These data suggested that DCD-tetraRFP had four DCD5 sterically arranged in different directions so that DCD-tetraRFP cross-linked with the substrate, causing aggregation. Lastly, the aggregates of DCD-tetraRFP and alpha-1,3-glucan captured Aspergillus oryzae conidia and decreased their biofilm formation by 80% in a 24-well dish. (C) 2022, The Society for Biotechnology, Japan. All rights reserved.
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Key words
α-1,3-Glucan-binding domain,Tetrameric red fluorescent protein,Fusion protein,Fungal cell wall,Fungal biofilm
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