HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplmental Research Data Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rice, J. J. Articles by Daugherty, P. S. Search for Related Content PubMed PubMed Citation Articles by Rice, J. J. Articles by Daugherty, P. S. Social Bookmarking                   What's this?

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

¹ Department of Chemical Engineering
² Institute for Collaborative Biotechnologies
³ Biomolecular Science and Engineering, University of California, Santa Barbara, California 93106, USA

(RECEIVED October 8, 2005; FINAL REVISION December 21, 2005; ACCEPTED December 22, 2005)

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/_YxEC, 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.

Keywords: bacterial display; peptide library; OmpX; streptavidin; high affinity ligand

Abbreviations: FACS, fluorescence-activated cell sortingGFP, green fluorescent proteinOMP, outer membrane proteinCPX, circularly permuted outer membrane protein OmpXYPet, yellow fluorescent protein for energy transferSA, streptavidinPE, R-phycoerythrinCm, Chloramphenicol

CiteULike

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. J. Rice and P. S. Daugherty Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides Protein Eng. Des. Sel., July 1, 2008; 21(7): 435 - 442. [Abstract] [Full Text] [PDF]

				J. Lofblom, J. Sandberg, H. Wernerus, and S. Stahl Evaluation of Staphylococcal Cell Surface Display and Flow Cytometry for Postselectional Characterization of Affinity Proteins in Combinatorial Protein Engineering Applications Appl. Envir. Microbiol., November 1, 2007; 73(21): 6714 - 6721. [Abstract] [Full Text] [PDF]

				K. T. Boulware and P. S. Daugherty Protease specificity determination by using cellular libraries of peptide substrates (CLiPS) PNAS, May 16, 2006; 103(20): 7583 - 7588. [Abstract] [Full Text] [PDF]