George P. Smith

Libraries of Peptides and Proteins Displayed on Filamentous Phage

Publisher Summary A fusion phage is a filamentous virion displaying on its surface a foreign peptide fused to a coat protein and harboring the gene for the fusion protein within its genome. This chapter emphasizes an application for which these surface expression vectors are particularly well suited: construction of epitope libraries. In such a library—the kind so far constructed—the phages display random foreign peptides encoded by degenerate synthetic oligonucleotides spliced into the coat protein gene, the library as a whole representing up to billions of peptide sequences. Affinity purified phages are eluted without destroying their infectivity, and the peptide sequences responsible for binding are easily ascertained by infecting the eluted phages into bacteria, propagating the resulting phage clones, and sequencing the relevant part of their viral DNAs. The number of peptides that can be accommodated with this technology exceeds by a factor of 100–1,000 the number that can be screened with conventional expression systems, in which the epitope is not displayed as part of the propagatable unit that encodes it. It also discusses more complex libraries, in which the displayed ligands are whole folded domains.

Surface presentation of protein epitopes using bacteriophage expression systems.

Vast libraries of filamentous phage expression vectors that display foreign (poly)peptides on the virion surface can be screened by affinity-purifying those phage whose displayed foreign peptide binds to an antibody or another binding protein. Present libraries display only short random peptides, but work is presently underway to create libraries displaying antibodies with a great diversity of binding specificities.

A new filamentous phage cloning vector: fd-tet

We have constructed a hybrid chromosome composed of the genome of wild-type fd (a filamentous, male-specific bacteriophage) and a segment of transposon Tn10 coding for tetracycline resistance but not including the Tn10 insertion sequences. The hybrid phage infects male E. coli, thereby transducing the infected cells to tetracycline resistance. The phage DNA can also be propagated in F- cells after transfection. This new phage, fd-tet, may be used as a cloning vector to produce large quantities of cloned DNA in single-stranded form. Its usefulness has been demonstrated by cloning of a fragment from bacteriophage lambda. Some unexpected sequence alterations have been identified in lambda cloning experiments.

Gene-III protein of filamentous phages: evidence for a carboxyl-terminal domain with a role in morphogenesis

A filamentous phage derivative, fCA55, bearing a nonpolar deletion in gene III, has been constructed and characterized to study the functions of that gene. The deletion eliminates most of gene III without disturbing its reading frame or the putative promoter for the downstream gene, VI. Therefore it is assumed that any abnormalities exhibited by fCA55 are a direct effect of the gene-III lesion itself, and not polar effects on other genes. fCA55 Is abnormal in two respects. First, it is noninfective; in this it resembles another nonpolar gene-III deletion mutant, fKN16, which is missing 507 bp encompassing roughly the first half of the gene. Second, it is secreted as polyphage--very long particles containing many unit-length DNA molecules; in this respect, fCA55 differs from fKN16. When the viral proteins of these two mutants were analyzed with antibody directed against gene-III protein, it was found that fKN16 contains an altered gene-III protein, while fCA55 is unreactive. It was concluded that the gene-III protein has two functional domains: the N-terminal domain, missing in both mutants, is required for viral infectivity; while the C-terminal domain, partly missing in fCA55 but retained in fKN16, is incorporated into the virion, and is responsible for the proteins role in generating normal, unit-length particles.

AFFINITY MATURATION OF PHAGE-DISPLAYED PEPTIDE LIGANDS

Publisher Summary When the fitness being selected for is affinity for a target receptor molecule, the foregoing program is called “affinity maturation,” the term coined by immunologists for the interspersed rounds of selective stimulation by antigen and somatic mutation of antibody genes that is thought to give rise to antibodies, with increasing affinity in the course of an immune response. This chapter discusses the affinity maturation from random peptide libraries displayed on phage. The procedures and underlying principles are discussed in the context of a specific exemplar experiment, in which ligands for a model receptor were selected from a library of random 15-mers. The model receptor is S-protein, a 104-residue fragment of bovine ribonuclease prepared by partial digestion, with subtilisin; the other fragment, S-peptide, corresponds to the N-terminal 20 amino acids. Neither fragment alone is enzymatically active, but when they are mixed S-peptide binds strongly to S-protein, restoring the enzyme activity.

Filamentous phage DNA cloning vectors: A noninfective mutant nonh a nonpolar deletion in gene III

Abstract A filamentous phage derivative, fKN16, has been constructed from the tetracyclineresistance transducing phage fd-tet by deleting a 507-base-pair (bp) segment of phage gene III. In accord with the importance of the gene III protein in infection, the infectivity of fKN16 phage is less than 10 −8 that of fd-tet phage. In contrast to most gene III amber mutants, which are polar on the downstream phage genes VI and I, fKN16 should be a nonpolar mutant since its 507-bp deletion spans an integral number of coding triplets. And indeed, two phage traits that may depend on gene VI and I function—the level of phage production and packaging into unit-length virions—appear to be normal in fKN16. High phage production coupled with very low infectivity make fKN16 suitable as a vector for DNA cloning experiments requiring a high level of biological containment. The characteristics of fKN16 as a vector were investigated in detail, using Hind III fragments of phage λ DNA as model foreign DNA. fKN16 may also be useful in studying the role of the gene III protein in the filamentous phage life cycle.

Identifying Diagnostic Peptides for Lyme Disease through Epitope Discovery

ABSTRACT Serum antibodies from patients with Lyme disease (LD) were used to affinity select peptide epitopes from 12 large random peptide libraries in phage display format. The selected peptides were surveyed for reactivity with a panel of positive sera (from LD patients) and negative sera (from subjects without LD), thus identifying 17 peptides with a diagnostically useful binding pattern: reactivity with at least three positive sera and no reactivity with any of the negative sera. The peptides define eight sequence motifs, none of which can be matched convincingly with segments of proteins from Borrelia burgdorferi, the LD pathogen; evidently, then, they are “mimotopes,” mimicking natural pathogen epitopes without matching contiguous amino acids of pathogen proteins. Peptides like these could be the basis of a new diagnostic enzyme-linked immunosorbent assay for LD, with sufficient specificity and sensitivity to replace expensive immunoblotting tests that are currently required for definitive serological diagnosis. Moreover, the method used to discover these peptides did not require any knowledge of the pathogen and involved generic procedures that are applicable to almost any infectious disease, including emerging diseases for which no pathogen has yet been identified.

Cloned mpc 11 myeloma cells express two kappa genes. A gene for a complete light chain and a gene for a constant region polypeptide.

Cloned MPC 11 mouse plasmacytoma cells synthesize a complete kappa light chain and also a kappa light chain constant region fragment. Partial amino terminal sequences of the in vitro forms of these two proteins have been determined. Both in vitro products contain typical light chain leaders; leaders are defined as the amino terminal sequences present on in vitro products but absent from the in vivo products found in living cells. The in vitro form of the MPC 11 complete light chain contains a leader sequence plus variable and constant region sequences. The in vitro form of the MPC 11 light chain constant region fragment contains a different leader sequence attached directly to a complete constant ragion sequence and has no variable region sequences. Thus the MPC 11 light chain fragment is not a degradation product of the MPC 11 complete light chain (or of any other complete light chain) and must be coded by a separate gene. The results reveal two unusual features of MPC 11 cells: first, expression of a unique variant light chain gene coding the light chain constant region fragment, and second, expression of two different kappa light chain genes (coding the complete light chain and the variant constant region fragment) in a single cell. In addition, evidence is provided that the in vitro forms of kappa light chains, three of which are presented here for the first time, include a minimum of three partially homologous but quite different leader sequences.

Immunogenically fit subunit vaccine components via epitope discovery from natural peptide libraries.

Antigenic peptides that bind pathogen-specific Abs are a potential source of subunit vaccine components. To be effective the peptides must be immunogenically fit: when used as immunogens they must elicit Abs that cross-react with native intact pathogen. In this study, antigenic peptides obtained from phage display libraries through epitope discovery were systematically examined for immunogenic fitness. Peptides selected from random peptide libraries, in which the phage-displayed peptides are encoded by synthetic degenerate oligonucleotides, had marginal immunogenic fitness. In contrast, 50% of the peptides selected from a natural peptide library, in which phage display segments of actual pathogen polypeptides, proved very successful. Epitope discovery from natural peptide libraries is a promising route to subunit vaccines.

Corruption of phage display libraries by target-unrelated clones: Diagnosis and countermeasures

Phage display is used to discover peptides or proteins with a desired target property-most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phages or peptides (TUPs), that lack the target behavior. Many TUPs are propagation related; they have mutations conferring a growth advantage and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus-strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus-strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus-strand origin. The founders infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption.