Display of various peptides and mini-proteins using eCPX (shaded) and CPX (white) as measured using flow cytometry

The -axis indicates the fold fluorescence above background for each protein target in the corresponding fluorescent channel. P2 was labeled with Mona, which is fused to the fluorescent protein YPet. CRPpep and V114 were labeled with biotinylated CRP and VEGF, respectively, then labeled with SA–PE. Mini-Z and T7pep were labeled with Alexa -conjugated human IgG and anti-T7•tag monoclonal IgG, respectively. SApep was detected with SA–PE.<p><b>Copyright information:</b></p><p>Taken from "Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides"</p><p></p><p>Protein Engineering, Design and Selection 2008;21(7):435-442.</p><p>Published online 13 May 2008</p><p>PMCID:PMC2427320.</p><p>© 2008 The Author(s)</p

Flow cytometric measurement of simultaneous N- and C-terminal display (bi-terminal display)

Overlays of two-parameter histograms resulting from clones displaying SApep and P2 on the N- and C-terminus, respectively, with a 6 () or a 26 () residue linker between SApep and the N-terminus of eCPX. In both (A) and (B) plots, the four distinct populations consist of non-displaying cells mock-labeled with SA–PE and Ypet-Mona (bottom left population), cells that display SApep and P2 labeled with only SA–PE (top left population), with SA–PE and YPet-Mona (top right population), or with only YPet-Mona (bottom right population).<p><b>Copyright information:</b></p><p>Taken from "Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides"</p><p></p><p>Protein Engineering, Design and Selection 2008;21(7):435-442.</p><p>Published online 13 May 2008</p><p>PMCID:PMC2427320.</p><p>© 2008 The Author(s)</p

Intracellular FRET-based Screen for Redesigning the Specificity of Secreted Proteases

Proteases are attractive as therapeutics given their ability to catalytically activate or inactivate their targets. However, therapeutic use of proteases is limited by insufficient substrate specificity, since off-target activity can induce undesired side-effects. In addition, few methods exist to enhance the activity and specificity of human proteases, analogous to methods for antibody engineering. Given this need, a general methodology termed protease evolution via cleavage of an intracellular substrate (PrECISE) was developed to enable engineering of human protease activity and specificity toward an arbitrary peptide target. PrECISE relies on coexpression of a protease and a peptide substrate exhibiting Förster resonance energy transfer (FRET) within the endoplasmic reticulum of yeast. Use of the FRET reporter substrate enabled screening large protease libraries using fluorescence activated cell sorting for the activity of interest. To evolve a human protease that selectively cleaves within the central hydrophobic core (KLVF↓F↓AED) of the amyloid beta (Aβ) peptide, PrECISE was applied to human kallikrein 7, a protease with Aβ cleavage activity but broad selectivity, with a strong preference for tyrosine (Y) at P1. This method yielded a protease variant which displayed up to 30-fold improvements in Aβ selectivity mediated by a reduction in activity toward substrates containing tyrosine. Additionally, the increased selectivity of the variant led to reduced toxicity toward PC12 neuronal-like cells and 16–1000-fold improved resistance to wild-type inhibitors. PrECISE thus provides a powerful high-throughput capability to redesign human proteases for therapeutic use

Design of a Cyclotide Antagonist of Neuropilin‑1 and -2 That Potently Inhibits Endothelial Cell Migration

Neuropilin-1 and -2 are critical regulators of angiogenesis, lymphangiogenesis, and cell survival as receptors for multiple growth factors. Disulfide-rich peptides that antagonize the growth factor receptors neuropilin-1 and neuropilin-2 were developed using bacterial display libraries. Peptide ligands specific for the VEGFA binding site on neuropilin-1 were identified by screening a library of disulfide-rich peptides derived from the thermostable, protease-resistant cyclotide kalata B1. First generation ligands were subjected to one cycle of affinity maturation to yield acyclic peptides with affinities of 40–60 nM and slow dissociation rate constants (∼1 × 10^–3 s^–1). Peptides exhibited equivalent affinities for human and mouse neuropilin-1 and cross-reacted with human neuropilin-2 with lower affinity. A C-to-N cyclized variant (cyclotide) of one neuropilin ligand retained high affinity, exhibited increased protease resistance, and conferred improved potency for inhibiting endothelial cell migration <i>in vitro</i> (EC₅₀ ≈ 100 nM). These results demonstrate that potent, target-specific cyclotides can be created by evolutionary design and that backbone cyclization can confer improved pharmacological properties

Antibody Repertoire Profiling Using Bacterial Display Identifies Reactivity Signatures of Celiac Disease

A general strategy to identify serum antibody specificities associated with a given disease state and peptide reagents for their detection was developed using bacterial display peptide libraries and multiparameter flow cytometry (MPFC). Using sera from patients with celiac disease (CD) (<i>n</i> = 45) or healthy subjects (<i>n</i> = 40), bacterial display libraries were screened for peptides that react specifically with antibodies from CD patients and not with those from healthy patients. The libraries were screened for peptides that simultaneously cross-react with CD patient antibodies present in two separate patient groups labeled with spectrally distinct fluorophores but do not react with unlabeled non-CD antibodies, thus affording a quantitative separation. A panel of six unique peptide sequences yielded 85% sensitivity and 91% specificity (AUC = 0.91) on a set of 60 samples not used for discovery, using leave-one-out cross-validation. Individual peptides were dissimilar with known CD-specific antigens tissue transglutaminase (tTG) and deamidated gliadin, and the classifier accuracy was independent of anti-tTG antibody titer. These results demonstrate that bacterial display/MPFC provides a highly effective tool for the unbiased discovery of disease-associated antibody specificities and peptide reagents for their detection that may have broad utility for diagnostic development

Validation of <i>BacKin</i> assay examined with flow cytometry.

<p>(A) Dotplot of side scatter and fluorescence emission intensity at 585/42 nm for eCPX-displaying cells and abltide-displaying cells. After treatment, the percentage of fluorescent cells (shown in green) is ∼0.1% in eCPX-displaying cells and > 96% in abltide-displaying cells. (B) Representative fluorescence intensity histograms for eCPX-displaying and abltide-displaying cells. The incubation time with kinase is given in brackets. All samples were fluorescently-labeled by incubation with biotin-anti-PY and SAPE. F is the mean fluorescence signal obtained for 100,000 cells analyzed.</p

Schematic representation of the Bcr-Abl tyrosine kinase.

<p>Tyrosine kinases, such as Bcr-Abl, are phosphoryl transferases that transfer phosphate from ATP to Tyr residues on specific substrate proteins. These enzymes have an ATP-binding site that is independent of the catalytic site; when bound to ATP, they become activated and exert their activity. (A) The catalytic and ATP-binding sites of Bcr-Abl are located in the Abl domain. (B) The 3D structure of the Abl domain with imatinib (red) bound to the ATP cleft (PDB: 3k5v); imatinib occludes the ATP binding and locks the enzyme in the inactive conformation.</p

<i>BacKin</i> assay procedure.

<p>(A) Map of plasmid pBAD33-eCPX-abltide (left) and schematic representation of abltide fused to eCPX expressed on the cell surface of an <i>E. coli</i> cell transformed with pBAD33-eCPX-abltide (right). The plasmid carries the arabinose promoter pBAD and resistance to chloramphenicol (CM^r). The recognition sites for the restriction enzyme SfiI and annealing region are show. (B) Schematic representation of the <i>BacKin</i> assay: 1. <i>E. coli</i> cells transformed with eCPX-substrate plasmid are incubated at 37°C and shaken to grow until mid log phase; 2. Substrate (e.g. abltide) expression and display on the bacteria surface is induced with arabinose; 3. Substrate phosphorylation, cells are incubated with kinase (e.g. Abl kinase) and an excess of ATP; 4. Phosphorylated substrate is labeled by incubation of cells with biotinylated-anti-phosphotyrosine antibody (biotin-anti-PY) followed by incubation with streptavidin-phycoerythrin (SAPE); 5. Mean fluorescence of kinase-treated cells is quantified by flow cytometry and compared with mean fluorescence of kinase untreated cells.</p

Characterization of Abl kinase using the <i>BacKin</i> assay.

<p>Mean fluorescence was converted into fraction of phosphorylation, assuming maximum response as 100% of abltide phosphorylation. Each data point is the average of three independent experiments±SD (A) Abltide phosphorylation catalyzed by increasing concentrations of Abl kinase in the presence of 500 µM ATP and incubated for 30 min at 37°C. The kinase concentration required to achieve half of the maximum response, K_D±SD, was calculated by fitting a nonlinear sigmoidal curve. (B) ATP concentration effect on abltide phosphorylation catalyzed by 0.5 U/mL Abl Kinase for 30 min at 37°C. The ATP concentration required to achieve half of the maximum response, K_D±SD, was calculated by fitting a nonlinear sigmoidal curve. (C) Time-course of abltide phosphorylation catalyzed by 0.5 U/mL Abl kinase in the presence of 500 µM ATP at 37°C obtained with <i>BacKin</i> assay (circles) or with LC-MS assay (squares). The time required to phosphorylate half of the abltide molecules, t_1/2±SD, was calculated by fitting a pseudo-first order association kinetics.</p

Kinase inhibition parameters.^[a]

[a]<p>Inhibitory concentration (IC₅₀) and Hill slope (H) determined by fitting dose-response plots with a sigmoidal curve (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080474#pone-0080474-g005" target="_blank">Figure 5</a>).</p