Rapid isolation of high-affinity protein binding peptides using bacterial display.
A robust bacterial display methodology was developed that allows the rapid isolation of peptides that bind to arbitrarily selected targets with high affinity. To demonstrate the utility of this approach, a large library (5 x 10(10) clones) was constructed composed of random 15-mer peptide insertions constrained within a flexible, surface exposed loop of the Escherichia coli outer membrane protein A (OmpA). The library was screened for binding to five unrelated proteins, including targets previously used in phage display selections: human serum albumin, anti-T7 epitope mAb, human C-reactive protein, HIV-1 GP120 and streptavidin. Two to four rounds of enrichment (2-4 days) were sufficient to enrich peptide ligands having high affinity for each of the target proteins. Strong amino acid consensus sequences were apparent for each of the targets tested, with up to seven consensus residues. Isolated peptide ligands remained functional when expressed as insertional fusions within a monomeric fluorescent protein. This bacterial display methodology provides an efficient process for identifying peptide affinity reagents and should be useful in a variety of molecular recognition applications.
Bessette
Paul H
PH
Department of Chemical Engineering, University of California, Santa Barbara, CA 93106, USA.
Rice
Jeffrey J
JJ
Daugherty
Patrick S
PS
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016384:Consensus Sequence; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D002352:Carrier Proteins; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins