Modular Approaches to Synthesize Activity- and Affinity-Based Chemical Probes

Combinatorial and modular methods to synthesize small molecule modulators of protein activity have proven to be powerful tools in the development of new drug-like molecules. Over the past decade, these methodologies have been adapted toward utilization in the development of activity- and affinity-based chemical probes, as well as in chemoproteomic profiling. In this review, we will discuss how methods like multicomponent reactions, DNA-encoded libraries, phage displays, and others provide new ways to rapidly screen novel chemical probes against proteins of interest.

Isolation and characterization of a novel bacteriophage, Kapi1, capable of O-antigen modification in commensal Escherichia coli.

In this study, we describe the isolation and characterization of novel bacteriophage Kapi1 (vB_EcoP_Kapi1) isolated from a strain of commensal Escherichia coli inhabiting the gastrointestinal tract of healthy mice. We show that Kapi1 is a temperate phage integrated into tRNA argW of strain MP1 and describe its genome annotation and structure. Kapi1 shows limited homology to other characterized prophages but is most similar to the phages of Shigella flexneri, and clusters taxonomically with P22-like phages. Investigation of the lifestyle of Kapi1 shows that this phage displays unstable lysogeny and influences the growth of its host. The receptor for Kapi1 is the lipopolysaccharide O-antigen, and we further show that Kapi1 alters the structure of its hosts O-antigen in multiple ways. We hope to use MP1 and Kapi1 as a model system to explore molecular mechanisms of mammalian colonization by E. coli and ask what the role(s) of prophages in this context might be.

Phage Display Selection of an Anti-Idiotype-Antibody with Broad-Specificity to Deoxynivalenol Mycotoxins

The use of synthetic antibody libraries and phage displays provides an efficient and robust method for the generation of antibodies against a wide range of targets with highly specific binding properties. As the in vitro selection conditions can be easily controlled, these methods enable the rapid generation of binders against difficult targets such as toxins and haptens. In this study, we used deoxynivalenol mycotoxin as a target to generate anti-idiotype-antibodies with unique binding properties from synthetic antibody libraries. The binding of the selected anti-idiotype antibodies can be efficiently inhibited with the addition of free isoforms of deoxynivalenol. The antibody was consecutively used to develop deoxynivalenol-specific ELISA and TRF-immunoassays, which can detect deoxynivalenol and two of the most common metabolic isoforms in the range of 78–115 ng/mL.

Complete Genome Sequence of Brucella abortus Phage EF4, Determined Using Long-Read Sequencing

Brucellaphage EF4 was isolated from elk feces. The 38,321-bp double-stranded DNA genome is predicted to contain 72 coding regions, 38 of which have been assigned predicted functions. This phage displays nucleotide similarity to other brucellaphages of the genus Perisivirus.

Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

Anatomy of a Lactococcal Phage Tail

ABSTRACT Bacteriophages of the Siphoviridae family utilize a long noncontractile tail to recognize, adsorb to, and inject DNA into their bacterial host. The tail anatomy of the archetypal Siphoviridae λ has been well studied, in contrast to phages infecting gram-positive bacteria. This report outlines a detailed anatomical description of a typical member of the Siphoviridae infecting a gram-positive bacterium. The tail superstructure of the lactococcal phage Tuc2009 was investigated using N-terminal protein sequencing, Western blotting, and immunogold transmission electron microscopy, allowing a tangible path to be followed from gene sequence through encoded protein to specific architectural structures on the Tuc2009 virion. This phage displays a striking parity with λ with respect to tail structure, which reenforced a model proposed for Tuc2009 tail architecture. Furthermore, comparisons with λ and other lactococcal phages allowed the specification of a number of genetic submodules likely to encode specific tail structures.

THE PREPARATION OF RELATIVELY PURE BACTERIOPHAGE

The method described above, based on the electrophoretic migration of bacteriophage particles into an agar gel and their subsequent re-suspension in a suitable medium, has the following advantages: It is simple and can be readily carried out on a comparatively large scale by merely inserting additional units between the same electrode cups. It requires but one extraction and the resulting phage suspension is strongly lytic, an average sample being capable of completely lysing susceptible bacteria at a dilution of 10–16. The suspension contains no proteins demonstrable by the biuret, alcohol, xanthoproteic, Millon or Hopkins-Cole reactions and yields but 0.044 mg. N/cc. directly attributable to the phage. Each corpuscle contains no more nitrogen than a single molecule of protein. In addition the method is applicable to determinations of the electric charge carried by biologically active substances of small dimensions, e.g., phage, toxins, and perhaps some viruses. It offers as well a possible means of purification of these substances. The purified bacteriophage obtained by such a procedure or similar ones is relatively unstable. Work now in progress indicates that it does not possess nearly the resistance to chemical agents, drying, etc., that non-purified phage displays. It is suggested that experiments designed to test the therapeutic value of bacteriophage be conducted, when possible, with purified suspensions thereby avoiding any possibility of obscure non-specific reactions due to other constituents of the lysates.