Jornal de Técnicas Analíticas e Bioanalíticas

Abstrato

Method for Discovery of Peptide Reagents Using a Commercial Magnetic Separation Platform and Bacterial Cell Surface Display Technology

Deborah A. Sarkes, Brandi L. Dorsey, Amethist S. Finch and Dimitra N. Stratis-Cullum

Biopanning by bacterial display has many advantages over yeast and phage display, including the speed to discovery of affinity reagents and direct amplification of bound cells without the need to elute and reinfect. However, widespread use is limited, in part due to poor performance achieved using manual Magnetic-Activated Cell Sorting (MACS) methods, and an absence of widely-available, low cost, high-performance sorting alternatives. Here, we have developed a methodology for bacterial cell sorting using the semi-automated autoMACS® Pro Separator for the first time, and have produced a complete method for sorting of bacteria displaying 15-mer peptides on their cell surface using this device, including downstream bioinformatic analysis of candidates for binding to a target of interest. Two autoMACS® programs designed for isolation of target cells with low frequency were evaluated and adapted to bacterial biopanning, using protective antigen (PA) of Bacillus anthracis as the model system. In contrast to manual MACS, the bacterial display library was preferentially enriched by autoMACS® sorting, yielding several promising candidates after only three rounds of biopanning and bioinformatic analysis. Individual candidates were evaluated for relative binding to fluorescently-labeled PA target or streptavidin negative control using Fluorescence-Activated Cell Sorting (FACS). The top thirteen peptide candidates from the autoMACS® sort demonstrate binding to PA with low cross-reactivity to streptavidin, while only two of eighteen candidates from the manual sort showed binding to PA, and both demonstrated greater cross-reactivity to streptavidin. Overall, the autoMACS® platform quickly harvested higher affinity peptide candidates with demonstrated specificity to the PA target. Peptide candidates produced with this method contained the previously reported PA consensus WXCFTC, further validating this method and the commercially available autoMACS® platform as the first low cost, semi-automated biopanning approach for bacterial display that is widely accessible and more reliable than the MACS/FACS standard protocol.