Screening of Peptide Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge

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

MACS/FACS results of MACS_545 Sample.

<p>Flow cytometry analysis of the fraction cell binding through conventional MACS plus FACS sorting after incubation with Streptavidin-Phycoerythrin (SAPE) labeled biotinylated PA protein target. The intensity of PE fluorescence (<i>y</i>-axis) represents either the expression of the surface peptide or affinity of the target to the display peptide. In the top dot plot, the cells are incubated with SAPE+biotin-PA prior to arabinose induction (negative control). The bottom dot plot shows the fraction PA bound after arabinose induction of the MACS_545 cells.</p

PA and Streptavidin binding analysis of Single Clones: The percentage binding from flow cytometry of ten of the MMS isolated clones and fourteen MACS/FACS isolated clones.

<p>Each clone was tested against Dylight 488 labeled PA at 150 nM and Streptavidin-Phycoerythrin (SAPE) at 150 nM to assess the initial affinity to PA and the cross-reactivity to SAPE at 150 nM.</p

Affinity Analysis of Single Clones.

<p>Flow cytometry affinity analysis of the highest affinity clones for MMS and the best MACS/FACS clone. The cells were analyzed for affinity using 500, 250, 50, 25, 5, 2.5, 0.5, and 0 nM of the PA-Dylight 488 sample. The dissociation constant (K_D) is shown for each sample.</p

MMS Disposable Cartridge.

<p>The disposable MMS cartridge is made from polypropylene with outer dimensions of 120 mm×55 mm. The sample is mixed with buffer 1 prior to entering the trapping region. Buffer 2 is used to wash away all of the cells not bound to magnetic beads in the trapping region, which are collected at the negative cell outlet. Buffer 2 is also used to elute the cells trapped on the magnetic beads into the positive cell outlet for overnight growth for subsequent sorting or analysis of the enrinched population.</p

Flow Cytometry Analysis of MMS Results per Sorting Round.

<p>Flow cytometry analysis of the fraction of target-binding clones in the enriched population after incubation with Streptavidin-R-Phycoerythrin (SAPE) fluorescently labeled biotinylated PA protein target. The intensity of PE fluorescence (<i>y</i>-axis) represents the level of binding on the cell surface; this may be due to either a high expression or a high affinity for the target. Following one round of MMS, 0.7% (net) of the population exhibited PA binding peptides. Following two rounds of MMS, 56.5% (net) of the population exhibited target-binding peptides. And 65.1% (net) of the population exhibited target-binding peptides after three rounds MMS selection.</p

Sequence Alignment of MMS and MACS/FACS Sorts.

<p>Peptide sequences of clones selected by MMS system and MACS for binding to protective antigen. Conserved residues are highlighted in blue, similar residues in green, and identical residues in yellow. All of the twenty-four MMS selected sequences and one of the MACS sequences contained a six residue consensus sequence of WXCFTC.</p

Rare-cell and Ultra-rare Cell Recovery Comparison.

<p>Rare-cell and ultra-rare cell recovery results for the MMS (black) and manual MACS (red) comparison. In four independent trials, both the MMS and MACS show cell recovery down to 10⁻⁸, with the MMS having greater between experiment cell recovery performance through all of the populations tested, given the smaller RMSD reported as error bars for each sample.</p