The Maltose-Binding Protein as a Scaffold for Monovalent Display of Peptides Derived from Phage Libraries

Abstract

Random peptide libraries are displayed on filamentous bacteriophage as fusions to either the minor coat protein, pIII, or the major coat protein, pVIII. We have devised a means of isolating the peptide displayed on a phage clone by transferring it to the N-terminus of the maltose-binding protein (MBP) of Escherichia coli encoded by malE. Transfer of a peptide sequence to monomeric MBP eliminates phage-encoded amino acids downstream of the insert peptide as well as avidity effects caused by multivalent display on phage. Peptide:MBP fusions are also easily affinity purified on amylose columns. The pMal-p2 vector was engineered to accept phage DNA encoding pIII-and pVIII-displayed peptides fused to their respective leader sequences. Both types of leader sequence were shown to target the peptide:MBP fusions to the periplasm of E. coli. A streamlined procedure for transferring peptides to MBP was applied to clones that had been isolated from a panel of pVIII-displayed peptide libraries by screening with an HIV-1-specific monoclonal antibody (Ab). By enzyme-linked immunosorbent assay, the Ab bound each of the peptide:MBP fusions and required the presence of a disulfide bridge within each peptide. Some of the peptide:MBP fusions were also analyzed using surface plasmon resonance. Thus, our study shows the value of malE fusion vectors in characterizing phage-displayed peptides.Phage display continues to grow as a technology for selecting peptides that bind to a variety of biomolecules including (Abs), 2 enzymes, receptors, DNA, and RNA (for reviews, see Refs. 1 and 2). The two phage proteins used for peptide display are the minor coat protein, pIII, and the major coat protein, pVIII. pIII is present in four or five copies that are closely © 1998 by Academic Press All rights of reproduction in any form reserved. 1To whom correspondence should be addressed. Fax: (604) 291-5583. jkscott@sfu.ca. 2 Abbreviations used: MBP, maltose-binding protein; Ab, antibody; MAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; SPR, surface plasmon resonance; DTT, dithiothreitol; BSA, bovine serum albumin; NANP, NANPNVDP(NANP) 3 GGPA; PBS, phophate-buffered saline; TBS, Tris-buffered saline; IPTG, isopropyl-thio-β-D-galactoside; RF, replicative form. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript clustered at one tip of the virion, whereas pVIII is present in thousands of copies that are arranged in a fish-scale-like pattern forming the body of the virion. Thus, multivalency can complicate the determination of the affinity of a selecting molecule for its cognate peptide displayed on either pIII or pVIII. In pIII display, avidity effects are produced by the close clustering of peptides. In pVIII display, the potential avidity effects vary between clones, as there is variation in the level of incorporation of recombinant peptide:pVIII fusions into the hybrid virion coat; this appears to be governed by the rate of processing of the pro-coat (3). Furthermore, in both cases th...

Human Immunodeficiency Virus Type 1-neutralizing Monoclonal Antibody 2F5 is Multispecific for Sequences Flanking the DKW Core Epitope

“…antigens immobilized on the chip and injected antibody) was not explored, since it has been described as generating unreliable data, due to the avidity effect inherent in bivalent molecules and the re-binding events that take place during the dissociation phase. 30,31 Thus, even though this experimental design is widely used for the determination of on and off rates, kinetic data generated with this procedure may be questionable. We favored the alternative of determining the affinity at equilibrium in solution as described by Nieba et al 30 In this method, the antibody is at a fixed concentration and is reacted to equilibrium with increasing antigen concentrations.…”

“…The generation of deletion mutants was performed using linear, single-stranded phage DNA as template, following the procedure described by Bonnycastle et al 27 Site-directed mutagenesis for the generation of amino acid substitutions and deletions were made with closed, circular single-stranded phage DNA as described by Sambrook et al 50 The transfer of peptide-coding sequences to the MBP fusion system pMALX and the conditions for culture and purification of the protein Multispecificity of HIV-1-Neutralizing Antibody 2F5 have been described by Zwick et al 31 DNA sequencing from partially purified phage clones was performed with the Thermo Sequenase II Dye Terminator Cycle kit (Amersham Biosciences, NJ) following the manufacture's instructions. DNA fragments from the sequencing reactions were resolved using an ABI 373 apparatus and analyzed with EditView 1.0.1 software.…”

Structure of a High-affinity “Mimotope” Peptide Bound to HIV-1-neutralizing Antibody b12 Explains its Inability to Elicit gp120 Cross-reactive Antibodies

“…The MBP-fusion protein was prepared as described, 51 and dimer was isolated and further purified by preparative SDS-PAGE as described by Castellanos-Serra et al 52 The affinity at equilibrium of IgG1 b12 for B2.1-MBP was measured using a KinExA 3000 instrument (Sapidyne Instruments, Boise, ID) as described, 53 and by SPR using a Biacore 3000 instrument (Biacore AB, Uppsala, Sweden; instrument located at the Laboratory of Molecular Biophysics, University of BC, Vancouver). Several methods for SPR analysis were used to validate the results.…”

Phage Display and Crystallographic Analysis Reveals Potential Substrate/Binding Site Interactions in the Protein Secretion Chaperone CsaA from Agrobacterium tumefaciens

“…Typically, recombinant pVIII molecules bearing peptide compose 0.5-10% of the total coat protein. 24 Three rounds of panning were performed, and the affinity of the pool selected in each cycle was analyzed by ELISA (data not shown). 25 Individual clones were amplified from these pools and showed significant binding in ELISA, above background binding for F88 phage.…”