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.

Figure 1

Nucleotide and amino acid sequences for constructs described in this paper. (A) pMal-pIII vector with pIII leader sequence, including KpnI(Acc65I) and EagI sites. (B) Ph.D. phage display library with random peptide sequence [X_n, (NNK)_n; K = G or T] followed by a GGGS linker, which is inserted between the pIII leader sequence and the mature protein. (C) pMal-X and pMal-KHP vectors with MBP:pVIII chimeric leader sequence with pVIII-derived residues in bold. (D) Phage-display vector f88.4 with pVIII leader sequence. (E) pVIII-displayed peptide library [X_n, (NNK)_n] in f88.4. Leader peptidase cleavage sites (^) are indicated.

Figure 2

PCR-subcloning strategy for transferring pVIII-displayed peptides from phage to MBP fusion vector pMal-X (see text for details).

Figure 3

Western blots of expression of MBP (A) produced by pMal-p2, and Erk1/2 MAP kinase-binding peptides N11 (B) and N15 (C) fused to MBP produced by pMal-pIII. Blots were probed with rabbit anti-MBP polyclonal Ab. (Lane 1) Whole-cell extract harvested immediately prior to IPTG induction or (lane 2) four hours after IPTG induction. (Lane 3) Osmotic shock supernatant. (Lane 4) resuspended osmotic shock pellet. P, precursor protein containing a leader sequence; M, mature processed protein without leader sequence.

Figure 4

Western blot of the G5:MBP fusion expressed by the vector pMal-X. The blot was first probed with rabbit anti-MBP polyclonal Ab (A) and then stripped and reprobed with MAb loop2 (B). (Lane 1) Whole-cell extract harvested immediately prior to IPTG induction or (lane 2) four hours after IPTG induction. (Lane 3) Osmotic-shock supernatant containing processed, G5:MBP protein (indicated by arrow). (Lane 4) Resuspended osmotic-shock pellet containing mostly unprocessed protein.

Figure 5

SDS–PAGE showing the effect of the prlA4 mutation on periplasmic levels of the C6:MBP fusion in E. coli. Osmotic-shock supernatants containing C6:MBP fusion protein were simultaneously produced in prlA⁺ DH5αF′ (lane 2) and AR182 (prlA4) cells (lane 3). The sizes (in kDa) of the molecular weight standards (lane 1) are indicated (left), as is the mature C6:MBP fusion protein (arrowhead, right).