Nanopore biosensing, a rapidly evolving bionanotechnology platform, utilizes protein pores for analyte translocation. The performance and selectivity of these biosensors are heavily influenced by the properties of its central component – the nanopore. The incorporation of non canonical amino acids (ncAAs) within the pore channel offers a powerful strategy for expanding the detectable analyte spectrum. However, incorporating ncAAs into recombinant proteins is a laborious and expensive process due to the production of truncated proteins and the burden on the host organism. To address this challenge, we have developed a high throughput screening assay for the efficient identification of functional variants of the pore-forming toxin (PFT) that contain ncAAs at different positions within the polypeptide chain. This assay utilizes minimal reagent volumes and allows the selection of variants that form functional pores suitable for biosensor applications. We employed lysenin and actinoporin M as model pore-forming toxins. Using molecular cloning techniques, we constructed fusion DNA sequences encoding PFT variants with a C-terminally fused green fluorescent protein (GFP). The screening assay involved monitoring the fluorescence of PFT fusion variants produced in E. coli in microtiter plates in vivo, followed by evaluation of their pore-forming ability using a hemolysis assay. Our results demonstrate that both the ncAA incorporation site and the specific ncAA type significantly impact the production level and functionality of the PFT variants. The successful production of wild-type model PFT nanopores validated the suitability of the constructed fusion DNA sequences for large-scale PFT and nanopore production. This high throughput ncAA incorporation screening assay serves as a valuable tool for assessing expression efficiency in small-volume cultures and optimizing ncAA incorporation conditions for diverse PFTs.
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