The maltose-binding protein as a scaffold for monovalent display of peptides derived from phage libraries.
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.
Biochemistry Program, Department of Biological Sciences, Institute of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, V5A 1S6, Canada.
M B
MB
Zwick
L L
LL
Bonnycastle
K A
KA
Noren
S
S
Venturini
E
E
Leong
C F
CF
Barbas
C J
CJ
Noren
J K
JK
Scott
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D015483:HIV Antibodies / Q000276:immunology; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D019151:Peptide Library; D033902:Periplasmic Binding Proteins; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D015483:HIV Antibodies; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D019151:Peptide Library; D033902:Periplasmic Binding Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli