Biology subjects

Imbert, L.

Publications and source records attributed to Imbert, L..

4 recordsLinked to original sources

bioRxiv2026

A modular platform for scalable recombinant production of highly toxic bacterial proteins

Bacterial protein toxins constitute a vast and largely untapped reservoir of antimicrobial activities with substantial therapeutic and biotechnological potential. However, their intrinsic toxicity frequently prevents stable recombinant expression in bacterial hosts, creating a major bottleneck for biochemical characterization, structural analysis, and development as antimicrobial agents. Here, we present a modular platform for the scalable recombinant production of highly toxic bacterial proteins based on transient intramolecular toxin neutralization. The strategy covalently links each toxin to its cognate immunity protein, promoting neutralization during biosynthesis while permitting recovery of the native toxin through site-specific proteolytic cleavage. Using this approach, we produced multiple previously intractable polymorphic toxin domains that could not be obtained using conventional inducible expression, toxin-immunity co-expression, or bacterial cell-free systems. We further streamlined the production workflow through intracellular protease-mediated cleavage, reducing the purification process from four steps to two and increasing protein recovery. To address cases in which native immunity proteins were insufficient, we incorporated computational protein design to engineer improved toxin-binding partners, enabling production of an additional toxin that remained refractory to the original platform. Purified toxins retained enzymatic activity following denaturation and refolding, confirming recovery of functional proteins and enabling identification of a previously uncharacterized nuclease activity. Together, these findings establish a scalable and adaptable microbial biotechnology platform for the production of intrinsically toxic proteins. The integration of transient intramolecular neutralization with computational engineering provides a route toward systematic production and characterization of toxic proteins for antimicrobial discovery, structural biology, protein engineering, and future biotechnological applications.

biochemistry↗

bioRxiv2025

A Fast and efficient strategy for the NMR assignment of Fab methyl groups

Owing to their high specificity and therapeutic effectiveness, monoclonal antibodies (mAbs) have rapidly become one of the leading classes of biologic drugs used to treat critical illnesses. The antigen-binding fragment (Fab) of mAbs plays a key role in the antigen recognition, so its structural characterization is essential, as even a slight change to its Higher Order Structure (HOS) can impact the antibodys potency. Recently, 2D methyl NMR has been introduced as a powerful method to assess both the structure and integrity of therapeutic Fab fragments. However, the identification of methyl group resonances in NMR spectra remains rare since Fabs are large heterodimers of [~]50 kDa. Here, we present the methyl group assignment of an IgG1 Fab produced in a cell-free system with an optimal isotope labelling. We first assigned 99% of the alanine, isoleucine, leucine, methionine, and valine methyl groups of the therapeutic Fab targeting LAMP1 antigen. Building on this assignment, we propose a "divide and conquer" strategy that exploits sequence identities to rapidly assign methyl groups of other IgG1 Fabs. We demonstrate that the assignment of the Fabs constant region can easily be transferred from one IgG1 to another and that the variable part of a new Fab can be assigned using smaller uniformly 15N,13C-labelled constructs. We applied our strategy to ipilimumabs Fab and, using the assignment of ipilimumabs variable part and Fab anti-LAMP1s constant part, we could transfer the assignment of 89% of the methyl-containing amino acids to the entire ipilimumab Fab without having to produce deuterated samples. This assignment strategy can be generalised to any other IgG1 Fabs provided that their constant regions are identical and the strategy can be adapted to accommodate the expression levels of the different variable domains. This new method drastically facilitates the Fab assignment process, making it suitable for the pharmaceutical timeline.

biochemistry↗

bioRxiv2023

Site-Specific Introduction of Alanines for the NMR Investigation of Low-Complexity Regions and Large Biomolecular Assemblies

NMR studies of large biomolecular machines and highly repetitive proteins remain challenging due to the difficulty of assigning signals to individual nuclei. Here, we present an efficient strategy to address this challenge by engineering a Pyrococcus horikoshii tRNA/alanyl-tRNA synthetase pair that enables the incorporation of up to three isotopically labeled alanine residues in a site-specific manner using in vitro protein expression. We have demonstrated the general applicability of this approach for NMR assignment by introducing isotopically labeled alanines into four proteins, including the 300-kDa molecular chaperone ClpP and the alanine-rich Phox2B transcription factor. For large protein assemblies, our labeling approach enables unambiguous assignments, while avoiding potential artefacts induced by site-specific mutations. When applied to Phox2B, which contains two poly-alanine tracts of nine and twenty alanines, we observe that the helical stability is strongly dependent on the homorepeat length, demonstrating structural cooperativity. The capacity to selectively introduce alanines with distinct labeling patterns is a powerful tool to probe structure and dynamics of biomolecular systems that are out of the reach of traditional structural biology methods.

biochemistry↗

bioRxiv2021

Determination of the Two-Component Systems regulatory network reveals core and accessory regulations across Pseudomonas aeruginosa lineages

Pseudomonas aeruginosa possesses one of the most complex bacterial regulatory networks, which largely contributes to its success as a human opportunistic pathogen. However, most of its transcription factors (TFs) are still uncharacterized and the potential intra-species variability in regulatory networks has been mostly ignored so far. Here, to provide a first global view of the two-component systems (TCSs) regulatory network in P. aeruginosa, we produced and purified all DNA-binding TCS response regulators (RRs) and used DAP-seq to map the genome-wide binding sites of these 55 TFs across the three major P. aeruginosa lineages. The resulting networks encompass about 40% of all genes in each strain and contain numerous new important regulatory interactions across most major physiological processes, including virulence and antibiotic resistance. Strikingly, the comparison between the three representative strains shows that about half of the detected targets are specific to only one or two of the tested strains, revealing a previously unknown large functional diversity of TFs within a single species. Three main mechanisms were found to drive this diversity, including differences in accessory genome content, as exemplified by the strain-specific plasmid in the IHMA87 outlier strain which harbors numerous binding sites of chromosomally-encoded RRs. Additionally, most RRs display potential auto-regulation or RR-RR cross-regulation, bringing to light the vast complexity of this network. Overall, we provide the first complete delineation of the TCS regulatory network in P. aeruginosa that will represent an important resource for future studies on this pathogen.

microbiology↗