HR-Bac, a toolbox based on homologous recombination for expression, screening and production of multiprotein complexes using the baculovirus expression system

International audienceAbstract The Baculovirus/insect cell expression system is a powerful technology for reconstitution of eukaryotic macromolecular assemblies. Most multigene expression platforms rely on Tn7-mediated transposition for transferring the expression cassette into the baculoviral genome. This allows a rigorous characterization of recombinant bacmids but involves multiple steps, a limitation when many constructs are to be tested. For parallel expression screening and potential high throughput applications, we have established an open source multigene-expression toolbox exploiting homologous recombination, thus reducing the recombinant baculovirus generation to a single-step procedure and shortening the time from cloning to protein production to 2 weeks. The HR-bac toolbox is composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. They contain single or dual expression cassettes bearing different affinity tags and their design facilitates the mix and match utilization of expression units from Multibac constructs. The overall cost of virus generation with HR-bac toolbox is relatively low as the preparation of linearized baculoviral DNA only requires standard reagents. Various multiprotein assemblies (nuclear hormone receptor heterodimers, the P-TEFb or the ternary CAK kinase complex associated with the XPD TFIIH subunit) are used as model systems to validate the toolbox presented

Inhibition of 11ß-hydroxysteroid dehydrogenase type 1 lowers intraocular pressure in patients with ocular hypertension

Background: Intraocular pressure (IOP) is maintained by a balance between aqueous humour (AH) production (dependent on sodium transport across a ciliary epithelial bi-layer) and drainage (predominantly through the trabecular meshwork). In peripheral epithelial tissues, sodium and water transport is regulated by corticosteroids and the 11ß-hydroxysteroid dehydrogenase (11ß-HSD) isozymes (11ß-HSD1 activating cortisol from cortisone, 11ß-HSD2 inactivating cortisol to cortisone). Aim: To analyse expression of 11ß-HSD in the human eye and investigate its putative role in AH formation. Design: Multipart prospective study, including a randomized controlled clinical trial. Methods: The expression of 11ß-HSD1 in normal human anterior segments was evaluated by in situ hybridization (ISH). RT-PCR for 11ß-HSDs, glucocorticoid and mineralocorticoid receptors (GR, MR) was performed on human ciliary body tissue. AH cortisol and cortisone concentrations were measured by radioimmunoassay on specimens taken from patients with primary open-angle glaucoma (POAG) and age-matched controls. Randomized, placebo-controlled studies of healthy volunteers and patients with ocular hypertension (OHT, raised IOP but no optic neuropathy) assessed the effect of oral carbenoxolone (CBX, an inhibitor of 11ß-HSD) on IOP. Results: ISH defined expression of 11ß-HSD1 in the ciliary epithelium, while RT-PCR analysis of ciliary body tissue confirmed expression of 11ß-HSD1, with additional GR and MR, but not 11ß-HSD2 expression. In both POAG patients and controls, AH concentrations of cortisol exceeded those of cortisone. The CBX-treated healthy volunteers who demonstrated the largest change in urinary cortisol metabolites, indicative of 11ß-HSD1 inhibition, had the greatest fall in IOP. Patients with OHT showed an overall reduction of IOP by 10% following CBX administration, compared to baseline (p&lt;0.0001). Discussion: CBX lowers IOP in patients with ocular hypertension. Our data suggest that this is mediated through inhibition of 11ß-HSD1 in the ciliary epithelium. Selective and topical inhibitors of 11ß-HSD1 could provide a novel treatment for patients with glaucoma. <br/

Active mRNA degradation by EXD2 nuclease elicits recovery of transcription after genotoxic stress

Here the authors show that the exonuclease EXD2 is involved in the recovery of class II gene transcription after UV irradiation. EXD2 travels from the mitochondria to the nucleus to interact with RNA Pol II and degrade new synthetized mRNA to allow transcription following DNA repair