Living Bacteria-Nanoparticle Hybrids Mediated through Surface-Displayed Peptides.

In this study, we investigated the preparation of living bacteria-nanoparticle hybrids mediated by surface-displayed peptides. The assembly of metallic nanoparticles on living bacteria has been achieved under mild conditions utilizing metal-peptide interactions, whereas the viability of the bacterial cells was greatly preserved. Escherichia coli was engineered with inducible gene circuits to control the display of peptides with desired sequences. Several designed peptide sequences as well as known gold-binding peptides were expressed on the cell surface using enhanced circularly permuted outer membrane protein X (eCPX) scaffolds. Driven by metal-peptide affinity, "biofriendly" citrate-stabilized gold nanoparticles were self-assembled onto the surface of bacteria with displayed peptides, which required overcoming the repulsive force between negatively charged nanoparticles and negatively charged cells. The bacteria/Au nanoparticle hybrids were highly viable and maintained the ability to grow and divide, which is a crucial step toward the creation of living material systems. Further activity and preservation of the bacterial hybrid assembly was demonstrated. The method described herein enables the conjugation of bacterial surfaces with diverse metal-rich nanoparticles in an inducible, and therefore easily controlled, manner. The expressed peptide sequences can be easily modified to alter the binding affinity and specificity for a wide variety of materials to form on-demand, high-density living biohybrids.

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

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.

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

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.

Isolation of cell specific peptide ligands using fluorescent bacterial display libraries.

Methods for identifying and producing cell specific affinity reagents are critical in cell detection, separation, and therapeutic delivery applications, yet remain difficult and time consuming. To address these limitations, a rapid and quantitative screening approach was developed using intrinsically fluorescent bacterial display peptide libraries and fluorescence-activated cell sorting (FACS). High-throughput screening of fluorescent libraries yielded a panel of peptide ligands mediating specific recognition of human breast cancer tumor cells. Clonal populations of fluorescent, peptide-displaying bacteria enabled single-step, fluorescent labeling of the target cells for cytometry and microscopy analysis. Isolated peptides could be categorized into several distinct groups possessing strong consensus sequences with as many as six identities. Importantly, individual clones exhibited high specificity target cell binding, with more than 80-fold increased binding to tumor cells (ZR-75-1) relative to cell lines derived from healthy tissue (HMEC, MCF-10A). Fluorescent display libraries thus provide a powerful new methodology for parallel identification of cell specific affinity ligands.

Rapid isolation of high-affinity protein binding peptides using bacterial display.

A robust bacterial display methodology was developed that allows the rapid isolation of peptides that bind to arbitrarily selected targets with high affinity. To demonstrate the utility of this approach, a large library (5 x 10(10) clones) was constructed composed of random 15-mer peptide insertions constrained within a flexible, surface exposed loop of the Escherichia coli outer membrane protein A (OmpA). The library was screened for binding to five unrelated proteins, including targets previously used in phage display selections: human serum albumin, anti-T7 epitope mAb, human C-reactive protein, HIV-1 GP120 and streptavidin. Two to four rounds of enrichment (2-4 days) were sufficient to enrich peptide ligands having high affinity for each of the target proteins. Strong amino acid consensus sequences were apparent for each of the targets tested, with up to seven consensus residues. Isolated peptide ligands remained functional when expressed as insertional fusions within a monomeric fluorescent protein. This bacterial display methodology provides an efficient process for identifying peptide affinity reagents and should be useful in a variety of molecular recognition applications.

Growth factors engineered for super-affinity to the extracellular matrix enhance tissue healing.

Growth factors (GFs) are critical in tissue repair, but their translation to clinical use has been modest. Physiologically, GF interactions with extracellular matrix (ECM) components facilitate localized and spatially regulated signaling; therefore, we reasoned that the lack of ECM binding in their clinically used forms could underlie the limited translation. We discovered that a domain in placenta growth factor-2 (PlGF-2(123-144)) binds exceptionally strongly and promiscuously to ECM proteins. By fusing this domain to the GFs vascular endothelial growth factor-A, platelet-derived growth factor-BB, and bone morphogenetic protein-2, we generated engineered GF variants with super-affinity to the ECM. These ECM super-affinity GFs induced repair in rodent models of chronic wounds and bone defects that was greatly enhanced as compared to treatment with the wild-type GFs, demonstrating that this approach may be useful in several regenerative medicine applications.

Tenascin C promiscuously binds growth factors via its fifth fibronectin type III-like domain.

Tenascin C (TNC) is an extracellular matrix protein that is upregulated during development as well as tissue remodeling. TNC is comprised of multiple independent folding domains, including 15 fibronectin type III-like (TNCIII) domains. The fifth TNCIII domain (TNCIII5) has previously been shown to bind heparin. Our group has shown that the heparin-binding fibronectin type III domains of fibronectin (FNIII), specifically FNIII12-14, possess affinity towards a large number of growth factors. Here, we show that TNCIII5 binds growth factors promiscuously and with high affinity. We produced recombinant fragments of TNC representing the first five TNCIII repeats (TNCIII1-5), as well as subdomains, including TNCIII5, to study interactions with various growth factors. Multiple growth factors of the platelet-derived growth factor (PDGF) family, the fibroblast growth factor (FGF) family, the transforming growth factor beta (TGF-ß) superfamily, the insulin-like growth factor binding proteins (IGF-BPs), and neurotrophins were found to bind with high affinity to this region of TNC, specifically to TNCIII5. Surface plasmon resonance was performed to analyze the kinetics of binding of TNCIII1-5 with TGF-ß1, PDGF-BB, NT-3, and FGF-2. The promiscuous yet high affinity of TNC for a wide array of growth factors, mediated mainly by TNCIII5, may play a role in multiple physiological and pathological processes involving TNC.

Engineering the regenerative microenvironment with biomaterials.

Modern synthetic biomaterials are being designed to integrate bioactive ligands within hydrogel scaffolds for cells to respond and assimilate within the matrix. These advanced biomaterials are only beginning to be used to simulate the complex spatio-temporal control of the natural healing microenvironment. With increasing understanding of the role of growth factors and cytokines and their interactions with components of the extracellular matrix, novel biomaterials are being developed that more closely mimic the natural healing environments of tissues, resulting in increased efficacy in applications of tissue repair and regeneration. Herein, the important aspects of the healing microenvironment, and how these features can be incorporated within innovative hydrogel scaffolds, are presented.

Protease-resistant peptide ligands from a knottin scaffold library.

Peptides within the knottin family have been shown to possess inherent stability, making them attractive scaffolds for the development of therapeutic and diagnostic agents. Given its remarkable stability to proteases, the cyclic peptide kalata B1 was employed as a scaffold to create a large knottin library displayed on the surface of E. coli. A library exceeding 10(9) variants was constructed by randomizing seven amino acids within a loop of the kalata B1 scaffold and screened using fluorescence-activated cell sorting to identify peptide ligands specific for the active site of human thrombin. Refolded thrombin binders exhibited high nanomolar affinities in solution and slow dissociation rates and were able to inhibit thrombin's enzymatic activity. Importantly, 80% of a knottin-based thrombin inhibitor remained intact after a 2 h incubation both with trypsin and with chymotrypsin, demonstrating that modifying the kalata B1 sequence did not compromise its stability properties. In addition, the knottin variant mediated 20-fold enhanced affinity for thrombin, when compared to the same seven residue binding epitope constrained by a single disulfide bond. Our results indicate that peptide libraries derived from the kalata B1 scaffold can yield high-affinity protein ligands that retain the remarkable protease resistance associated with the parent scaffold. More generally, this strategy may prove useful in the development of stable peptide ligands suitable for in vivo applications.