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.
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.
Lu
Z
Z
Genetics Institute, Inc, Cambridge, MA 02140, USA.
Murray
K S
KS
Van Cleave
V
V
LaVallie
E R
ER
Stahl
M L
ML
McCoy
J M
JM
D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics*; D005408:Flagellin / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics*
D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D005408:Flagellin; D013879:Thioredoxins