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Protein Sci ; 15(4): 825-36, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16600968

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.


Subject(s)
Bacterial Outer Membrane Proteins/isolation & purification , Escherichia coli Proteins/isolation & purification , Hydrolases/isolation & purification , Ligands , Membrane Proteins/chemistry , Peptide Library , Peptides/chemistry , Affinity Labels , Amino Acid Sequence , Antibody Affinity , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Humans , Hydrolases/genetics , Hydrolases/metabolism , Membrane Proteins/metabolism , Microscopy, Fluorescence , Models, Biological , Molecular Sequence Data , Peptides/isolation & purification , Peptides/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Streptavidin/chemistry , Streptavidin/metabolism
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