title: Screening of peptide libraries against protective antigen of Bacillus anthracis in a disposable microfluidic cartridge.
abstract: Bacterial surface peptide display has gained popularity as a method of affinity reagent generation for a wide variety of applications ranging from drug discovery to pathogen detection. In order to isolate the bacterial clones that express peptides with high affinities to the target molecule, multiple rounds of manual magnetic activated cell sorting (MACS) followed by multiple rounds of fluorescence activated cell sorting (FACS) are conventionally used. Although such manual methods are effective, alternative means of library screening which improve the reproducibility, reduce the cost, reduce cross contamination, and minimize exposure to hazardous target materials are highly desired for practical application. Toward this end, we report the first semi-automated system demonstrating the potential for screening bacterially displayed peptides using disposable microfluidic cartridges. The Micro-Magnetic Separation platform (MMS) is capable of screening a bacterial library containing 3 × 10¹⁰ members in 15 minutes and requires minimal operator training. Using this system, we report the isolation of twenty-four distinct peptide ligands that bind to the protective antigen (PA) of Bacilus anthracis in three rounds of selection. A consensus motif WXCFTC was found using the MMS and was also found in one of the PA binders isolated by the conventional MACS/FACS approach. We compared MMS and MACS rare cell recovery over cell populations ranging from 0.1% to 0.0000001% and found that both magnetic sorting methods could recover cells down to 0.0000001% initial cell population, with the MMS having overall lower standard deviation of cell recovery. We believe the MMS system offers a compelling approach towards highly efficient, semi-automated screening of molecular libraries that is at least equal to manual magnetic sorting methods and produced, for the first time, 15-mer peptide binders to PA protein that exhibit better affinity and specificity than peptides isolated using conventional MACS/FACS.
authors.affiliation: United States Army Research Laboratory, Sensors and Electron Devices Directorate, Adelphi, Maryland, United States of America.
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Kogot
authors.forename: Yanting
authors.initials: Y
authors.lastname: Zhang
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Moore
authors.forename: Paul
authors.initials: P
authors.lastname: Pagano
authors.forename: Dimitra N
authors.initials: DN
authors.lastname: Stratis-Cullum
authors.forename: David
authors.initials: D
authors.lastname: Chang-Yen
authors.forename: Marek
authors.initials: M
authors.lastname: Turewicz
authors.forename: Paul M
authors.initials: PM
authors.lastname: Pellegrino
authors.forename: Andre
authors.initials: A
authors.lastname: de Fusco
authors.forename: H Tom
authors.initials: HT
authors.lastname: Soh
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Stagliano
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology*; D001408:Bacillus anthracis / Q000276:immunology*; D001427:Bacterial Toxins / Q000737:chemistry / Q000276:immunology*; D004209:Disposable Equipment; D005434:Flow Cytometry; D008280:Magnetics; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods*; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000032:analysis* / Q000276:immunology; D011485:Protein Binding; D016415:Sequence Alignment; D019809:Streptavidin / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D019151:Peptide Library; D010455:Peptides; C030325:anthrax toxin; D019809:Streptavidin