Ubiquitous carbohydrate binding modules decorate 936 lactococcal siphophage virions

With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins, as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural “bricks” to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches

High-throughput production of oxidized animal toxins in Escherichia coli

High-throughput production (HTP) of synthetic genes is becoming an important tool to explore the biological function of the extensive genomic and meta-genomic information currently available from various sources. One such source is animal venom, which contains thousands of novel bioactive peptides with potential uses as novel therapeutics to treat a plethora of diseases as well as in environmentally benign bioinsecticide formulations. Here, we describe a HTP platform for recombinant bacterial production of oxidized disulfide-rich proteins and peptides from animal venoms. High-throughput, host-optimized, gene synthesis and subcloning, combined with robust HTP expression and purification protocols, generate a semiautomated pipeline for the accelerated production of proteins and peptides identified from genomic or transcriptomic libraries. The platform has been applied to the production of thousands of animal venom peptide toxins for the purposes of drug discovery, but has the power to be universally applicable for high-level production of various and diverse target proteins in soluble form. This chapter details the HTP protocol for gene synthesis and production, which supported high levels of peptide expression in the E. coli periplasm using a cleavable DsbC fusion. Finally, target proteins and peptides are purified using automated HTP methods, before undergoing quality control and screening

High-Throughput Production of a New Library of Human Single and Tandem PDZ Domains Allows Quantitative PDZ-Peptide Interaction Screening Through High-Throughput Holdup Assay

International audiencePDZ domains recognize PDZ Binding Motifs (PBMs) at the extreme C-terminus of their partner proteins. The human proteome contains 266 identified PDZ domains, the PDZome, spread over 152 proteins. We previously developed the "holdup" chromatographic assay for high-throughput determination of PDZ-PBM affinities. In that work, we had used an expression library of 241 PDZ constructs (the "PDZome V.1"). Here, we cloned, produced, and characterized a new bacterial expression library ("PDZome V.2"), which comprises all the 266 known human PDZ domains as well as 37 PDZ tandem constructs. To ensure the best expression level, folding, and solubility, all construct boundaries were redesigned using available structural data and all DNA sequences were optimized for Escherichia coli expression. Consequently, all the PDZ constructs are produced in a soluble form. Precise quantification and quality control were carried out. The binding profiles previously published using "PDZome V.1" were reproduced and completed using the novel "PDZome V.2" library. We provide here the detailed description of the high-throughput protocols followed through the PDZ gene synthesis and cloning, PDZ production, holdup assay and data treatment

High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery

International audienceAnimal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane receptors. Disulphide-reticulated peptides have evolved to display improved specificity, low immunogenicity and to show much higher resistance to degradation than linear peptides. These properties make venom peptides attractive candidates for drug development. However, recombinant expression of reticulated peptides containing disulphide bonds is challenging, especially when associated with the production of large libraries of bioactive molecules for drug screening. To date, as an alternative to artificial synthetic chemical libraries, no comprehensive recombinant libraries of natural venom peptides are accessible for high-throughput screening to identify novel therapeutics

Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli

Abstract Background Animal venoms are large, complex libraries of bioactive, disulphide-rich peptides. These peptides, and their novel biological activities, are of increasing pharmacological and therapeutic importance. However, recombinant expression of venom peptides in Escherichia coli remains difficult due to the significant number of cysteine residues requiring effective post-translational processing. There is also an urgent need to develop high-throughput recombinant protocols applicable to the production of reticulated peptides to enable efficient screening of their drug potential. Here, a comprehensive study was developed to investigate how synthetic gene design, choice of fusion tag, compartment of expression, tag removal conditions and protease recognition site affect levels of solubility of oxidized venom peptides produced in E. coli . Results The data revealed that expression of venom peptides imposes significant pressure on cysteine codon selection. DsbC was the best fusion tag for venom peptide expression, in particular when the fusion was directed to the bacterial periplasm. While the redox activity of DsbC was not essential to maximize expression of recombinant fusion proteins, redox activity did lead to higher levels of correctly folded target peptides. With the exception of proline, the canonical TEV protease recognition site tolerated all other residues at its C-terminus, confirming that no non-native residues, which might affect activity, need to be incorporated at the N-terminus of recombinant peptides for tag removal. Conclusions This study reveals that E. coli is a convenient heterologous host for the expression of soluble and functional venom peptides. Using the optimal construct design, a large and diverse range of animal venom peptides were produced in the \ub5M scale. These results open up new possibilities for the high-throughput production of recombinant disulphide-rich peptides in E. coli

Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms

International audienceSeptin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9. Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9 thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled the first-time isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin/microtubule cytoskeleton