Enrichment Of Phages Research Articles

Selective enrichment and characterization of high affinity ligands from collections of random peptides on filamentous phage

Large collections of random peptides can be expressed on the N-terminus of the pIII protein of filamentous phage and screened for binding to antibodies and other receptors. In our previous work with a monoclonal antibody (3E7) (Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378–6382, 1990), we showed that a high proportion of the selected peptides had relatively low affinity ( K d 's > 1 μm). Here we describe conditions for selective enrichment of phage expressing high affinity peptides. This is done by allowing the phage to interact with a low concentration of 3E7 Fab followed by extensive washing to allow dissociation of phage-bearing peptides with low affinity. These affinity selection conditions were applied to the pool of phage previously selected using a high concentration of IgG. A phage clone with the known high affinity ligand YGGFL ( K d 7.1 n m) and several other closely related peptides were isolated. The dissociation rate of 125I-3E7 Fab from several phage clones approximated that of phage expressing YGGFL. A good correlation was found between the dissociation rate of the peptides found on phage and the equilibrium binding constants of chemically synthesized peptides. The strategy of using a low concentration of receptor and extensive washing to select phagebearing high affinity peptides, combined with assays to determine the specificity and relative affinity of peptides on isolated phage clones, should be generally applicable in using the peptides-on-phage system for discovery of high affinity receptor ligands.

Making antibody fragments using phage display libraries

To by-pass hybridoma technology and animal immunization, we are trying to build antibodies in bacteria by mimicking features of immune selection. Recently we used fd phage to display antibody fragments fused to a minor coat protein, allowing enrichment of phage with antigen. Using a random combinatorial library of the rearranged heavy (VH) and kappa (V kappa) light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx), we have now displayed diverse libraries of antibody fragments on the surface of fd phage. After a single pass over a hapten affinity column, fd phage with a range of phOx binding activities were detected, at least one with high affinity (dissociation constant, Kd = 10(-8) M). A second pass enriched for the strong binders at the expense of the weak. The binders were encoded by V genes similar to those found in anti-phOx hybridomas but in promiscuous combinations (where the same V gene is found with several different partners). By combining a promiscuous VH or V kappa gene with diverse repertoires of partners to create hierarchical libraries, we elicited many more pairings with strong binding activities. Phage display offers new ways of making antibodies from V-gene libraries, altering V-domain pairings and selecting for antibodies with good affinities.

A Bacteriophage-Typing System for Surveying the Diversity and Distribution of Strains ofErwinia carotovorain Potato Fields

A bacteriophage-typing system was developed and used to survey the diversity and distribution among strains of Erwinia carotovora subsp. carotovora and E. c. subsp. atroseptica from the rhizospheres and stems of potatoes grown in the Columbia Basin of the Pacific Northwest. With a phage enrichment method and strains of E. carotovora from 25 serogroups as hosts, 13 phages displaying distinct host-range activities were isolated from potato and soil samples. In addition, a potato strain of E. chrysanthemi was used to isolate phage N (Ech-3), which did not infect strains of E. carotovora (...)

Populations of bacteriophage infecting Halobacterium in a transient brine pool

Phage enrichment cultures with Halobacterium cuturibrum as host, prepared from samples of diminishing volume collected from a tropical eurhaline brine pool, revealed that phages of relatively low virulence predominated in the phage population when both phage and host populations varied considerably in density. The largest number of phages observed occurred after destruction of the host population by rainfall. The results are discussed with respect to possible implications for phage ecology and evolution.

Populations of bacteriophage infectingHalobacteriumin a transient brine pool

Phage enrichment cultures with Halobacterium cuturibrum as host, prepared from samples of diminishing volume collected from a tropical eurhaline brine pool, revealed that phages of relatively low virulence predominated in the phage population when both phage and host populations varied considerably in density. The largest number of phages observed occurred after destruction of the host population by rainfall. The results are discussed with respect to possible implications for phage ecology and evolution.

Physico-chemical properties of filamentous phage *f2 of Xanthomonas campestirs pv. oryzae.

The physical properties and chemical structures of the filamentous phage Xf2 of Xanthomonas campestris pv. oryzae were clarified. The phage Xf2 was concentrated from enriched phage suspensin by differential centrifugation, purified by CsCl equilibrium density-gradient centrifugation and used for chemical analysis. The phage Xf2 was found to be composed of about 11% DNA and 89% of protein. The base composition of Xf2 DNA was adenine: guanine: cytosine: thymine=22.7:30.4:25.8:21.1, indicating high similarity to that of the X. campestris pv. oryzae phage Xf. Xf2 DNA was characterized as single-stranded one because of its noncomplementary base ratios and melting curve. SDS polyacrylamide gel electrophoresis proved that Xf2 protein consisted of major and minor proteins. Major coat protein was found to consist of 43 amino acid residues, showing some differences from that of Xf and filamentous coliphages. Histidine, cystine and phenylalanie were absent and the content of hydrophobic amino acids was calculated at 15 molecules per one coat protein. The molecular weight of major coat protein of Xf2 determined by amino acid composition analysis was 4740 daltons, and that of minor coat protein determined by gel electrophoresis was 56, 000 daltons.

Mapping of a gene for a major outer membrane protein ofEscherichia coli K12 with the aid of a newly isolated bacteriophage

A method is described for the enrichment of phages which can adsorb to a specific determinant of bacterial cell surfaces. A phage was isolated which absorbs to E. coli cells containing the "major outer membrane= protein c but not to strains that are lacking this protein. With the aid of this phage a gene, meoA which is responsible for the lack of protein c was mapped at 48 min on the linkage map of E. coli K12.

錦江湾のバクテリオファージに関する研究

The isolation and the purification of bacteriophages living in the coastal sea waters of Kinko Bay were studied. The characteristics of phages compared with those of host bacteria were also studied. The authors found that in order to isolate the phage from sea water, the enrichment of phages before isolation is indispensable. The characteristics of isolated phages were as follows; 1. Six bacteriophages were isolated from the sea waters of Kinko Bay. 2. Among the six host bacteria, two were halophilic and one of them was pseudomonas species, but the remaining were not classifiable according to BAIN and SHEWAN'S classification. Of six isolates four species were classified as marine bacteria. According to BAIN and SHEWAN'S classification, two of the marine bacteria were Pseudomonas and the other two were Vibrios. 3. As compared with host bacteria. All bacteriophages isolated demonstrated strong resistance to various agents; i.e., to chemicals, to hypotonic solutions such as diluted artificial sea water.

Isolation and preliminary characterization of bacteriophages for Bacillus subtilis.

Romig, W. R. (University of California, Los Angeles), and A. M. Brodetsky. Isolation and preliminary characterization of bacteriophages for Bacillus subtilis. J. Bacteriol. 82:135-141. 1961.-A simplified procedure for direct isolation of phages for Bacillus subtilis from soil was developed. Phage enrichment was accomplished by growing streptomycin-resistant B. subtilis in medium previously inoculated with the soil sample. Contaminating soil bacteria were eliminated by adding bactericidal quantities of streptomycin and the phages were isolated by conventional agar layer techniques. By this method 1 or more subtilis phages were isolated from 15 of 18 soil samples tested. Several of these phages were unusually sensitive to chloroform and all of them were relatively unstable when stored at refrigerator temperatures. Of 6 phages retained for study, 1 was temperate for B. subtilis, but attempts to obtain stably lysogenic bacteria following infection with this phage were unsuccessful. All 6 phages had identical host ranges and were able to lyse all strains of B. subtilis tested, as well as several related species of Bacillus.