Biomedical subjects

Jeffrey J Rice

Publications and source records attributed to Jeffrey J Rice.

3 recordsLinked to original sources

PubMed2006

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.

Affinity Labels↗

PubMed2006

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.

Affinity Labels↗

PubMed2004

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.

Amino Acid Sequence↗