Heterologous expression and secretion of nanobodies targeting Campylobacter jejuni for intestinal health applications
Date
2016
Authors
McLean, Richard
University of Lethbridge. Faculty of Arts and Science
Journal Title
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Publisher
Lethbridge, Alta : University of Lethbridge, Dept. of Chemistry and Biochemistry
Abstract
As strategies for engineering the enteric microflora continue to advance, the vision of a future with a secondary, artificial immune system increasingly comes into focus. There are numerous challenges that will need to be overcome before this is a reality including the construction of a chassis capable of producing functional antimicrobial compounds at adequate concentrations and the construction of libraries of antimicrobial compounds capable of targeting a range of pathogens including those that develop resistance.
Towards these ends, I have elected to engineer Bacteroides thetaiotaomicron (B. theta), one of the most prevalent and stable organisms in the human distal gut, to heterologously express and secrete nanobodies that bind the flagella of Campylobacter jejuni. Nanobody genes were inserted behind native B. theta promoters and integrated into the genome allowing for induction following the introduction of a specific inducing compound. Signalling peptides were fused to the nanobodies allowing for targeting the nanobodies out of Escherichia coli (E. coli) cells. Also as part of this project, novel signal peptides have also been characterized allowing for the targeting of protein to any subcellular compartment within a gram-negative bacterium.
Description
Keywords
Bacteroides , Campylobacter , nanobody , artificial immune systems , enteric engineering , flagella binding