ABSTRACT
Random peptide libraries displayed on the surface of filamentous bacteriophage are widely used as tools for the discovery of ligands for biologically relevant macromolecules, including antibodies, enzymes, and cell surface receptors. Phage display results in linkage of an affinity-selectable function (the displayed peptide) to the DNA encoding that function, allowing selection of individual binding clones by iterative cycles of in vitro panning and in vivo amplification. Critical to the success of a panning experiment is the complexity of the library: the greater the diversity of clones within the library, the more likely the library contains sequences that will bind a given target with useful affinity. A method for construction of high-complexity (> or = 10(9) independent clones) random peptide libraries is presented. The key steps are highly efficient binary ligation under conditions where the vector is relatively dilute, with only a modest molar excess of insert, followed by efficient electrotransformation into Escherichia coli. Library design strategies and a protocol for rapid sequence characterization are also presented.
Subject(s)
DNA/chemistry , Genetic Techniques , Peptide Library , Amino Acid Sequence , Base Sequence , Epitope Mapping/methods , Escherichia coli , Gene Library , Kinetics , Molecular Sequence Data , Plasmids/metabolism , TransfectionABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters , Carrier Proteins/genetics , Escherichia coli Proteins , Monosaccharide Transport Proteins , Peptide Library , Periplasmic Binding Proteins , Amino Acid Sequence , Bacteriophages/genetics , Base Sequence , Carrier Proteins/metabolism , Cloning, Molecular , DNA, Recombinant , Escherichia coli , Genetic Vectors , HIV Antibodies/immunology , HIV-1/immunology , Humans , Maltose-Binding Proteins , Molecular Sequence Data , Polymerase Chain Reaction , Protein Processing, Post-Translational , Protein Sorting Signals/genetics , Protein Sorting Signals/metabolism , Recombinant Fusion Proteins/geneticsABSTRACT
An amber suppressor tRNA was prepared in vitro by runoff transcription with T7 RNA polymerase. Both full-length tRNA and truncated tRNA lacking the 3' terminal pCpA from the acceptor stem could be synthesized from the same DNA template. Truncated runoff suppressor tRNA could be enzymatically ligated to phenylalanyl-pCpA to generate aminoacylated full-length suppressor tRNA (Phe-tRNA(CUA)). Phe-tRNA(CUA) is capable of suppressing an amber (UAG) mutation in vitro with equivalent efficiency as suppressor prepared by anticodon-loop replacement of a naturally-isolated tRNA. The ease of suppressor tRNA preparation using this method, compared to anticodon-loop replacement, greatly facilitates the use of chemically acylated suppressor tRNA's for site-specifically incorporating unnatural amino acids into proteins.