Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides

Abstract

Bacterial cell-surface display systems coupled with quantitative screening methods offer the potential to expand protein engineering capabilities. To more fully exploit this potential, a unique bacterial surface display scaffold was engineered to display peptides more efficiently from the surface exposed C- and N-termini of a circularly permuted outer membrane protein. Using directed evolution, efficient membrane localization of a circularly permuted OmpX (CPX) display scaffold was rescued, thereby improving the presentation of diverse passenger peptides on the cell surface. Random and targeted mutagenesis directed towards linkers joining the native N- and C-termini of OmpX coupled with screening by FACS yielded an enhanced CPX (eCPX) variant which localized to the outer membrane as efficiently as the non-permuted parent. Interestingly, enhancing substitutions coincided with a C-terminal motif conserved in outer membrane proteins. Surface localization of various passenger peptides and mini-proteins was expedited using eCPX relative to that achieved with the parent scaffold. The new variant also permitted simultaneous display and labeling of distinct peptides on structurally adjacent C- and N-termini, thus enabling display level normalization during library screening and the display of bidentate or dimeric peptides. Consequently, the evolved scaffold, eCPX, expands the range of applications for bacterial display. Finally, this approach provides a route to improve the performance of cell-surface display vectors for protein engineering and design.

Fig. 1

Display of a streptavidin-binding peptide using OmpX, CPX and eCPX. Flow cytometric analysis of E. coli displaying a streptavidin-binding peptide (AECHPQGPPCIEGRK) (Giebel et al., 1995) as an insertion into OmpX, an N-terminal fusion to CPX or an N-terminal fusion to eCPX after varying durations of induction. The cells were induced at room temperature for time increments between 0 and 90 min then labeled with 100 nM SA–PE.

Fig. 2

Locations of enhancing mutations within eCPX. Image was created using Molecular Operating Environment using the reported structure of OmpX (Vogt and Schulz, 1999), joining the N- and C-termini with the six residue linker GSKSRR, and creating the new termini within the second extracellular loop. Enhancing substitutions A165L and G166S are shown as space filling residues.

Fig. 3

Display of various peptides and mini-proteins using eCPX (shaded) and CPX (white) as measured using flow cytometry. The X-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 Alexa⁴⁸⁸ -conjugated human IgG and anti-T7•tag monoclonal IgG, respectively. SApep was detected with SA–PE.

Fig. 4

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 (A) or a 26 (B) 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).