One-step generation of error-prone PCR libraries using Gateway® technology

<p>Abstract</p> <p>Background</p> <p>Error-prone PCR (epPCR) libraries are one of the tools used in directed evolution. The Gateway^®technology allows constructing epPCR libraries virtually devoid of any background (<it>i.e</it>., of insert-free plasmid), but requires two steps: the BP and the LR reactions and the associated <it>E. coli </it>cell transformations and plasmid purifications.</p> <p>Results</p> <p>We describe a method for making epPCR libraries in Gateway^®plasmids using an LR reaction without intermediate BP reaction. We also describe a BP-free and LR-free sub-cloning method for in-frame transferring the coding sequence of selected clones from the plasmid used to screen the library to another one devoid of tag used for screening (such as the green fluorescent protein). We report preliminary results of a directed evolution program using this method.</p> <p>Conclusions</p> <p>The one-step method enables producing epPCR libraries of as high complexity and quality as does the regular, two-step, protocol for half the amount of work. In addition, it contributes to preserve the original complexity of the epPCR product.</p

Split-GFP Reassembly Assay: Strengths and Caveats from a Multiparametric Analysis

The split-Green Fluorescent Protein (GFP) reassembly assay is a powerful approach to study protein-protein interactions (PPIs). In this assay, two proteins, respectively, fused to the first seven and the last four β-strands of GFP are co-expressed in E. coli where they can bind to each other, which reconstitutes the full-length GFP. Thus, the fluorescence of the bacteria co-expressing the two fusion proteins accounts for the interaction of the two proteins of interest. The first split-GFP reassembly assay was devised in the early 2000s in Regan's lab. During the last ten years, we have been extensively using this assay to study the interactions of an intrinsically disordered protein (IDP) with two globular partners. Over that period, in addition to accumulating molecular information on the specific interactions under study, we progressively modified the original technique and tested various parameters. In those previous studies, however, we focused on the mechanistic insights provided by the approach, rather than on the method itself. Since methodological aspects deserve attention and the best bipartite reporter to study PPIs involving IDPs remains to be identified, we herein focus on technical aspects. To this end, we first revisit our previous modifications of the original method and then investigate the impact of a panel of additional parameters. The present study unveiled a few critical parameters that deserve consideration to avoid pitfalls and obtain reliable results

Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris

International audienceFilamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to setup the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community

FOXA1 mutations alter pioneering activity, differentiation and prostate cancer phenotypes.

Mutations in the transcription factor FOXA1 define a unique subset of prostate cancers but the functional consequences of these mutations and whether they confer gain or loss of function is unknown1-9. Here, by annotating the landscape of FOXA1 mutations from 3,086 human prostate cancers, we define two hotspots in the forkhead domain: Wing2 (around 50% of all mutations) and the highly conserved DNA-contact residue R219 (around 5% of all mutations). Wing2 mutations are detected in adenocarcinomas at all stages, whereas R219 mutations are enriched in metastatic tumours with neuroendocrine histology. Interrogation of the biological properties of wild-type FOXA1 and fourteen FOXA1 mutants reveals gain of function in mouse prostate organoid proliferation assays. Twelve of these mutants, as well as wild-type FOXA1, promoted an exaggerated pro-luminal differentiation program, whereas two different R219 mutants blocked luminal differentiation and activated a mesenchymal and neuroendocrine transcriptional program. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) of wild-type FOXA1 and representative Wing2 and R219 mutants revealed marked, mutant-specific changes in open chromatin at thousands of genomic loci and exposed sites of FOXA1 binding and associated increases in gene expression. Of note, ATAC-seq peaks in cells expressing R219 mutants lacked the canonical core FOXA1-binding motifs (GTAAAC/T) but were enriched for a related, non-canonical motif (GTAAAG/A), which was preferentially activated by R219-mutant FOXA1 in reporter assays. Thus, FOXA1 mutations alter its pioneering function and perturb normal luminal epithelial differentiation programs, providing further support for the role of lineage plasticity in cancer progression

PROTEINES INTRINSEQUEMENT DESORDONNEES : DE LA MUTAGENÈSE ALÉATOIRE À LA RELATION STRUCTURE-FONCTION

Les protéines intrinsèquement désordonnées (PID) sont dépourvues de structures secondaire et tertiaire en conditions physiologiques. La plupart des PID sont impliquées dans des fonctions cellulaires intervenant dans des étapes clés de la vie cellulaire, telles que la régulation du cycle cellulaire, la transduction d’un signal et la transcription. Une caractéristique notable des PID est leur capacité à interagir avec de multiples partenaires tout en étant spécifiques. Bien que cette propriété semble liée à leur flexibilité structurale, aucune étude n’a été entreprise jusqu’à présent qui permettrait de comprendre les déterminants moléculaires de la reconnaisance de partenaire.Le but de ce projet est de tenter de comprendre et de préciser les bases moléculaires déterminant l’affinité et la spécificité des PID lors de la reconnaissance de leur(s) partenaire(s). Pour cela, l’approche in vitro utilisée a consisté en la génération de mutants aléatoires au sein de la partie C-terminale de la nucléoprotéine NTAIL (domaine désordonné) du virus de la rougeole. Le domaine NTAIL subit un repliement induit en _-hélice au niveau de la région d’interaction Box2 (aa 489-506), lors de sa liaison avec son partenaire naturel et structuré PXD (partie C-terminale de la Phosphoprotéine). Le système de criblage utilisé est fondé sur le réassemblage de la Green Fluorescent Protein (GFP), où NTAIL et PXD sont chacun fusionnés à une moitié de la GFP. Ainsi, l’affinité de l’interaction des variants de NTAIL pour PXD, guide la reconstitution de la GFP.La mesure de la fluorescence des variants de NTAIL, corrélée à l’analyse de leur séquence peptidique, a permis de confirmer l’implication de certains résidus de la Box2 dans la liaison avec le partenaire. De plus, cette étude met en évidence que la pré-configuration en hélice de la région Box2 facilite l’interaction. Enfin, grâce à cette approche d’évolution descriptive, nous avons identifié des sites de contact primaire, en dehors de la Box2, capables de favoriser l’interaction entre NTAIL et son partenaire PXD

Compaction and binding properties of the intrinsically disordered C-terminal domain of Henipavirus nucleoprotein as unveiled by deletion studies

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Dissecting Partner Recognition by an Intrinsically Disordered Protein Using Descriptive Random Mutagenesis

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Order and Disorder in the Replicative Complex of Paramyxoviruses

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