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

链霉亲和素 融合蛋白 肽库 分子生物学 蛋白质标签 噬菌体展示 生物 计算生物学 化学 肽序列 生物化学 生物素 重组DNA 基因
作者
Jeffrey J. Rice,Aaron Schohn,Paul Bessette,Kevin T. Boulware,Patrick S. Daugherty
出处
期刊:Protein Science [Wiley]
卷期号:15 (4): 825-836 被引量:110
标识
DOI:10.1110/ps.051897806
摘要

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.
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