Isolation of Predation-Deficient Mutants of Bdellovibrio bacteriovorus by Using Transposon Mutagenesis▿

The availability of the complete genome sequence of Bdellovibrio bacteriovorus provides an opportunity for investigating genes that play a significant role in predation. Using two independently derived facultatively predatory Bdellovibrio strains, we have designed a method to cultivate and screen transposon insertion mutants in 96-well microtiter dishes. Transposon insertion mutants were produced by introducing the plasposon pRL27, which carries a mini-Tn5. Mutants have been screened for predatory activity using 96-well plates. Seventeen independent nonpredatory mutants have been isolated, and DNA flanking the insertion has been sequenced. BLAST analysis revealed that most of these interrupted DNA sequences do not code for known proteins or functions. Two of the inactivated genes were analyzed further: one was found to code for a putative serine protease and the other a probable protein involved in secretion through the outer membrane. The methods described here are the first for the generation and isolation of predation-deficient mutants using random-transposon-insertion mutagenesis. As more mutants are isolated and their gene products analyzed, more light will be shed on how this predator carries out its exclusive life processes and perhaps how these products, or the organism itself, can be used for therapeutic, agricultural, and/or other purposes

Kinetic mechanism of controlled Fab-arm exchange for the formation of bispecific immunoglobulin G1 antibodies

Bispecific antibodies (bsAbs) combine the antigen specificities of two distinct Abs and demonstrate therapeutic promise based on novel mechanisms of action. Among the many platforms for creating bsAbs, controlled Fab-arm exchange (cFAE) has proven useful based on minimal changes to native Ab structure and the simplicity with which bsAbs can be formed from two parental Abs. Despite a published protocol for cFAE and its widespread use in the pharmaceutical industry, the reaction mechanism has not been determined. Knowledge of the mechanism could lead to improved yields of bsAb at faster rates as well as foster adoption of process control. In this work, a combination of Forster resonance energy transfer (FRET), nonreducing SDS-PAGE, and strategic mutation of the Ab hinge region was employed to identify and characterize the individual steps of cFAE. Fluorescence correlation spectroscopy (FCS) was used to determine the affinity of parental (homodimer) and bispecific (heterodimer) interactions within the C(H)3 domain, further clarifying the thermodynamic basis for bsAb formation. The result is a clear sequence of events with rate constants that vary with experimental conditions, where dissociation of the K409R parental Ab into half-Ab controls the rate of the reactio

A stable, engineered TL1A ligand co-stimulates T cells via specific binding to DR3

Abstract TL1A (TNFSF15) is a TNF superfamily ligand which can bind the TNFRSF member death receptor 3 (DR3) on T cells and the soluble decoy receptor DcR3. Engagement of DR3 on CD4+ or CD8+ effector T cells by TL1A induces downstream signaling, leading to proliferation and an increase in secretion of inflammatory cytokines. We designed a stable recombinant TL1A molecule that (1) displays high monodispersity and stability, (2) displays the ability to activate T cells in vitro and in vivo, and (3) lacks binding to DcR3 while retaining functional activity via DR3. Together these results suggest the TL1A ligand can be amenable to therapeutic development on its own or paired with a tumor-targeting moiety