Molecular cloning, expression and characterization of two potential antibacterial fusion hydrophobins in Pichia pastoris

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Title: Molecular cloning, expression and characterization of two potential antibacterial fusion hydrophobins in Pichia pastoris
Author: Jiang, Xinyu
Contributor: University of Helsinki, Faculty of Agriculture and Forestry, Department of Food and Environmental Sciences
Publisher: Helsingin yliopisto
Date: 2015
Language: eng
Thesis level: master's thesis
Discipline: Livsmedelskemi
Food Chemistry
Abstract: Biofilms constitute a successful protection mechanism for planktonic bacterial cells to survive in hostile environments. To date, biofilm-associated infections in medical devices represent a major cause of morbidity and mortality among patients. As a potential candidate for anti-biofilm therapy, fungal hydrophobins provide new solutions to manipulate the physical and chemical properties of surfaces, which in turn may give protection against bacterial colonization. However, in practice, native hydrophobin coatings generally have no impact on bacterial surface colonization, because of the lack of being antibacterial by these fungal proteins themselves. The aim of this study was to explore the feasibility of using recombinant fusion hydrophobins to control bacterial growth. In this study, the class I hydrophobin hgfI gene isolated from the edible mushroom Grifola frondosa was in frame fused with two antimicrobial peptide genes (bac8c and p11-5), respectively, and subsequently cloned into the corresponding expression vectors with a view to obtain two recombinant fusion hydrophobins, Bac8c-HGFI and P11-5-linker-HGFI. These two chimeric genes were separately expressed in Pichia pastoris under the regulation of alcohol oxidase 1 promoter. SDS-PAGE and immunoblot analyses confirmed that these two fusion proteins were successfully expressed and secreted into the culture medium. Minimum inhibitory concentration (MIC) test demonstrated that the highly active antimicrobial peptide Bac8c became inactivated when it was fused with the hydrophobin HGFI. Interestingly, the hydrophobin HGFI gained an acquired antibacterial nature when it was fused with the antimicrobial peptide P11-5 through a 10-mer flexible polypeptide linker, with the MIC of 100 μg/ml against Escherichia coli. To the best of my knowledge, this study presents the first heterologous expression of an antibacterial fusion hydrophobin in P. pastoris. This finding in combination with surface modification mediated by hydrophobin may broaden the current approaches used for anti-biofilm therapies.
Subject: antimicrobial peptide
fusion expression
polypeptide linker
Pichia pastoris

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