Expression of Thioredoxin Random Peptide Libraries on the Escherichia coli Cell Surface as Functional Fusions to Flagellin: A System Designed for Exploring Protein-Protein Interactions

Abstract

We have developed a system for probing protein/protein interactions which makes use of the bacterial flagellum to display random peptide libraries on the surface of E. coli. In developing the system the entire coding sequence of E. coli thioredoxin (trxA) was inserted into a dispensable region of the gene for flagellin (fliC), the major structural component of the E. coli flagellum. The resulting fusion protein (FLITRX) was efficiently exported and assembled into partially functional flagella on the bacterial cell surface. A diverse library of random dodecapeptides were displayed in FLITRX on the exterior of E. coli as conformationally constrained insertions into the thioredoxin active-site loop, a location known to be a highly permissive site for the insertion of exogenous peptide sequences into native thioredoxin. To demonstrate that members of this library could be bound and selected via specific protein/protein interactions to a target protein, a method was devised to enable efficient isolation of those bacteria displaying peptides with affinity to immobilized antibodies. We have unambiguously mapped three different antibody epitopes using this method. Peptides selected as FLITRX active-site fusions retain their binding specificity when made as native thioredoxin active-site loop fusions. This will facilitate future structural characterizations and broaden the general utility of the system for exploring other classes of protein-protein interactions.

A Protein Phosphatase-1-binding Motif Identified by the Panning of a Random Peptide Display Library

“…Growth of the E. coli Peptide Library-The FliTrx random peptide library was obtained from Invitrogen (San Diego, CA) and is based on the system described by Lu et al (28). Growth of the cultures and general panning methods were essentially as described in the manufacturer's protocol or as by Lu et al (28).…”

“…Because of the basic nature of these peptides, it is possible that this inhibition is similar to the inhibition of phosphorylase phosphatase activity by polyamines, which has previously been shown to be likely a substrate-directed effect (33). DISCUSSION In this study, we have used a random peptide display library in which peptides are inserted into a surface loop at the active site of thioredoxin (31), which itself is fused into a nonessential domain of the bacterial flagellin protein (28). Our results clearly demonstrate that a short peptide sequence containing the general motif VXF or VXW preceded by 1 or more basic residues is sufficient to generate a capacity for the fusion proteins to bind to PP1.…”

Isolation of peptide aptamers that inhibit intracellular processes

“…Similar constructions have been used to make thioredoxin fusions that have been selected for several biological activities (3,16,18). A 16-aa random peptide has approximately 6.6 ϫ 10 20 possible sequences.…”

Production of recombinant proteins in Escherichia coli

“…Display of peptides genetically fused to flagellin can be attained after introduction of heterologous sequences into a cloned flagellin gene expressed in bacterial strains devoid of a chromosomally-encoded structural subunit but proficient in all other genes required for flagellar expression, processing and assembly. One particularly interesting expression system based on E. coli flagellin relies on the insertion of thioredoxin into a central hypervariable surface-exposed flagellin domain (Lu et al, 1995). Thioredoxin represents by itself a versatile scaffold for display of fused peptides at conformations compatible with binding to other peptides and fusion with flagellin targets the hybrid protein to the cell surface.…”