Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands

Abstract

A bacterial display methodology was developed for N- and C-terminal display and demonstrated to enable rapid screening of very large peptide libraries with high precision and efficiency. To overcome limitations of insertional fusion display libraries, a new scaffold was developed through circular permutation of the Escherichia coli outer membrane protein OmpX that presents both N and C termini on the external cell surface. Circularly permuted OmpX (CPX) display was directly compared to insertional fusion display by screening comparable peptide libraries in each format using magnetic and fluorescence activated cell sorting. CPX display enabled in situ measurement of dissociation rate constants with improved accuracy and, consequently, improved affinity discrimination during screening and ranking of isolated clones. Using streptavidin as a model target, bacterial display yielded the well-characterized HP(Q)/(M) motif obtained previously using several alternative peptide display systems, as well as three additional motifs (L(I)/(V) CQNVCY, CGWMY(F)/(Y)xEC, ERCWYVMHWPCNA). Using CPX display, a very high affinity streptavidin-binding peptide was isolated having a dissociation rate constant k(off) = 0.002sec(-1) even after grafting to the C terminus of an unrelated protein. Comparison of individual clones obtained from insertional fusion and terminal fusion libraries suggests that the N-terminal display yields sequences with greater diversity, affinity, and modularity. CPX bacterial display thus provides a highly effective method for screening peptide libraries to rapidly generate ligands with high affinity and specificity.

Figure 1

Diagram of bacterial display scaffolds OmpX and CPX. (A) Structure of outer membrane protein OmpX (Vogt and Schulz 1999) with extracellular loops L2 and L3 projecting beyond the lipopolysac charide (LPS) layer, alongside a topological depiction of OmpX and CPX, where the native OmpX N and C termini are fused together by a GGSG linker (not to scale), and the newly formed N and C termini reside on the cell surface. (B) Gene map of OmpX and CPX depicting the araBAD promoter and ribosomal binding site as an arrow, followed by the OmpX signal sequence, and the remainder of the gene and stop codon. The vertical lines depict the portion of the gene encoding A1-S53, and the diagonal lines are the remaining codons. The linking sequences are shown as black lines, the asymmetric SfiI sites are indicated with subscripts, and the wild-type residues are labeled.

Figure 2

Display of a SA-binding peptide using OmpX or CPX. Flow cytometric analysis of E. coli displaying a SA binding peptide (RLEICQNVCYYLGTL) (Bessette et al. 2004) as an insertion into OmpX (A) or an N-terminal fusion to CPX (B), after varying durations of induction. The cells were induced at room temperature for time increments between 0 and 180 min, then labeled with 100 nM SA-PE.

Figure 3

Fluorescence microscopy analysis of peptide surface localization using CPX display. Bacterial cells coexpressing GFP and CPX₁-S1 (A) or bacterial cells coexpressing GFP and CPX (no peptide) (B) were labeled with streptavidin-conjugated quantum dots. Images were acquired using GFP and PE filter sets and then overlaid.

Figure 4

Enrichment of streptavidin-binding clones from a CPX library as measured using flow cytometry. Flow cytometric analysis after labeling with 5 nM SA-PE revealed 0.01% SA-PE positive cells prior to selection (A), 1% after two rounds of MACS (B), and 85% after two rounds of MACS and one round of FACS (C). Plots use a four-decade log scale.

Figure 5

Dissociation rate constants of selected clones measured on the cell surface or for soluble proteins. (A) Measurement of the dissociation rate of several streptavidin binding bacterial clones after labeling with SA-PE and addition of biotin as a competitor. (Diamonds) CX7₁-S1, (triangles) OX7-S1, (circles) CX7₂-S2, (asterisks) OX7-S2, (squares) CX7₂-SA3. (B) Measurement of SA binding peptide–YFP fusions bound to SA-conjugated polymeric beads after addition of biotin as a competitor. (Diamonds) CX7₁-S1, (circles) CX7₂-S2, (triangles) OX7-S1b.

Figure 6

Specificity of isolated clones for streptavidin. The extent of peptide binding to unrelated proteins, including anti-T7 tag monoclonal antibody at 200 nM, human serum albumin (HSA) at 500 nM, C-reactive protein (CRP) at 500 nM, and the Mona/Gads SH3 domain at 500 nM, as measured using flow cytometry.