Establishment and characterization of a novel secretion system in the methylotrophic yeast Pichia pastoris

Die methylotrophe Hefe Pichia pastoris gilt als beliebtes Expressionssystem zur Produktion von rekombinanten Proteinen. Die Verfügbarkeit sowohl von starken Promotoren (z.B. der Glyceraldehyd-3-Phosphat Dehydrogenase Promoter, PGAP und den Promoter der Alkohol Oxidase, PAOX1) und effizienten Sekretionssignalen (alpha mating factor Sekretionssignal; MF1αpp), als auch eine funktionierende Proteinfaltung und das Anfügen von post-translationalen Modifikationen sind vorteilhaft. Das extrazelluläre Protein X (Epx1) wurde als stark sekretiertes, jedoch unbekanntes Protein im Überstand von P. pastoris bemerkt. Wenig ist bekannt über Epx1, außer dass es eines von 20 endogen sekretierten Proteinen ist und der C-Terminus mit einer SCP-ähnlichen (sekretiert und Cystein-reich) extrazellulären Proteindomäne überein stimmt. Die Anwendbarkeit als neues Sekretionssystem für rekombinante Proteine wurde anhand von intrazellulärer eGFP Expression durch den Promoter (1.000 bp oberhalb des ATG) auf drei unterschiedlichen Kohlenstoffquellen getestet. PEPX1 zeigte keine verbesserte eGFP Sekretion, verglichen mit PGAP. Weiters wurde das Epx1 Sekretionssignal mit drei strukturell und funktionell unterschiedlichen Proteinen analysiert (pTRP, porcines Trypsinogen; HSA, humanes Serumalbumin; eGFP, verbessertes grün-fluoreszierendes Protein). Dadurch wurde eine neue und effiziente Sekretionssequenz für die Produktion rekombinanter Proteine etabliert.The methylotrophic yeast Pichia pastoris is successfully used for recombinant protein production. The application of strong promoters (e.g. the glyceraldehyde-3-phosphate dehydrogenase promoter PGAP, and the alcohol oxidase promoter, PAOX) and efficient secretion signals (alpha mating factor secretion leader; MFα1pp) as well as proper folding and post- translational modifications are beneficial. The extracellular protein X (Epx1) was detected as unknown, strongly secreted host protein in supernatants. As one of 20 endogenous secreted proteins in P. pastoris, little is known about Epx1 except C-terminal alignment with the SCP-like (secretory cysteine rich) extracellular protein domain. Therefore, the practicability as novel secretion system for enhancing recombinant protein expression was tested. The promoter sequence (1,000 bp upstream of ATG) was used to analyze intracellular eGFP expression. The Epx1 promoter, tested at three different carbon sources, was weaker in expressing eGFP compared to PGAP. Furthermore, secretion and processing mediated by Epx1 secretion leader was studied on three structurally and functionally different recombinant proteins: pTRP (porcine trypsinogen), HSA (human serum albumin), and eGFP (enhanced green fluorescent protein). Thereby we established a novel and efficient secretion leader system for the production of recombinant proteins

Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum

Background: Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector® micro-fermentation system. Results: Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that PlacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. PxylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. PlacSynth was inducible with TMG (methyl -D-thiogalactopyranoside) and IPTG (isopropyl -D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter PlacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector® micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a constitutive promoter). Conclusions: We evaluated different inducible promoters, as well as an orthologous expression system, for controlled gene expression in L. plantarum. Furthermore, here we provide proof of concept for a T7 RNA polymerase based expression system for L. plantarum. Thereby we expanded the molecular toolbox for an industrial relevant and generally regarded as safe (GRAS) strain.(VLID)101641

Status Report of the DPHEP Study Group: Towards a Global Effort for Sustainable Data Preservation in High Energy Physics

Data from high-energy physics (HEP) experiments are collected with significant financial and human effort and are mostly unique. An inter-experimental study group on HEP data preservation and long-term analysis was convened as a panel of the International Committee for Future Accelerators (ICFA). The group was formed by large collider-based experiments and investigated the technical and organisational aspects of HEP data preservation. An intermediate report was released in November 2009 addressing the general issues of data preservation in HEP. This paper includes and extends the intermediate report. It provides an analysis of the research case for data preservation and a detailed description of the various projects at experiment, laboratory and international levels. In addition, the paper provides a concrete proposal for an international organisation in charge of the data management and policies in high-energy physics

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Examining the impact of acetylene on N-fixation and the active sediment microbial community

Here we examined the impact of a commonly employed method used to measure nitrogen fixation, the acetylene reduction assay (ARA), on a marine sediment community. Historically, the ARA technique has been broadly employed for its ease of use, in spite of numerous known artifacts. To gauge the severity of these effects in a natural environment, we employed high-throughput sequencing to detect differences in acetylene-treated sediments versus non-treated control sediments after a seven hour incubation. Within this short time period, significant differences were seen broadly across all types of microbes identified in the sediment, implying that the changes induced by acetylene occur quickly. The results have important implications for our understanding of marine nitrogen budgets. Moreover, because the ARA technique has been widely used in terrestrial and freshwater habitats, these results may be applicable to other ecosystems

Proteomic and Transcriptomic Elucidation of the Mutant Ralstonia eutropha G+1 with Regard to Glucose Utilization▿ †

By taking advantage of the available genome sequence of Ralstonia eutropha H16, glucose uptake in the UV-generated glucose-utilizing mutant R. eutropha G+1 was investigated by transcriptomic and proteomic analyses. Data revealed clear evidence that glucose is transported by a usually N-acetylglucosamine-specific phosphotransferase system (PTS)-type transport system, which in this mutant is probably overexpressed due to a derepression of the encoding nag operon by an identified insertion mutation in gene H16_A0310 (nagR). Furthermore, a missense mutation in nagE (membrane component EIICB), which yields a substitution of an alanine by threonine in NagE and may additionally increase glucose uptake, was identified. Phosphorylation of glucose is subsequently mediated by NagF (cytosolic PTS component EIIA-HPr-EI) or glucokinase (GlK), respectively. The inability of the defined deletion mutant R. eutropha G+1 ΔnagFEC to utilize glucose strongly confirms this finding. In addition, secondary effects of glucose, which is now intracellularly available as a carbon source, on the metabolism of the mutant cells in the stationary growth phase occurred: intracellular glucose degradation is stimulated by the stronger expression of enzymes involved in the 2-keto-3-deoxygluconate 6-phosphate (KDPG) pathway and in subsequent reactions yielding pyruvate. The intermediate phosphoenolpyruvate (PEP) in turn supports further glucose uptake by the Nag PTS. Pyruvate is then decarboxylated by the pyruvate dehydrogenase multienzyme complex to acetyl coenzyme A (acetyl-CoA), which is directed to poly(3-hydroxybutyrate). The polyester is then synthesized to a greater extent, as also indicated by the upregulation of various enzymes of poly-β-hydroxybutyrate (PHB) metabolism. The larger amounts of NADPH required for PHB synthesis are delivered by significantly increased quantities of proton-translocating NAD(P) transhydrogenases. The current study successfully combined transcriptomic and proteomic investigations to unravel the phenotype of this hitherto-undefined glucose-utilizing mutant

Crystallization of Calcium Oxalates Is Controlled by Molecular Hydrophilicity and Specific Polyanion-Crystal Interactions

To gain more insight into protein structure-function relationships that govern ectopic biomineralization processes in kidney stone formation, we have studied the ability of urinary proteins (Tamm-Horsfall protein, osteopontin (OPN), prothrombin fragment 1 (PTF1), bikunin, lysozyme, albumin, fetuin-A), and model compounds (a bikunin fragment, recombinant-, milk-, bone osteopontin, poly-L-aspartic acid (poly asp), poly-L-glutamic acid (poly glu)) in modulating precipitation reactions of kidney stone-related calcium oxalate mono- and dihydrates (COM, COD). Combining scanning confocal microscopy and fluorescence imaging, we determined the crystal faces of COM with which these polypeptides interact; using scanning electron microscopy, we characterized their effects on crystal habits and precipitated volumes. Our findings demonstrate that polypeptide adsorption to COM crystals is dictated first by the polypeptide\u27s affinity for the crystal followed by its preference for a crystal face: basic and relatively hydrophobic macromolecules show no adsorption, while acidic and more hydrophilic polypeptides adsorb either nonspecifically to all faces of COM or preferentially to {100}/{121} edges and {100} faces. However, investigating calcium oxalates grown in the presence of these polypeptides showed that some acidic proteins that adsorb to crystals do not affect crystallization, even if present in excess of physiological concentrations. These proteins (albumin, bikunin, PTF1, recombinant OPN) have estimated total hydrophilicities from 200 to 850 kJ/mol and net negative charges from -9 to -35, perhaps representing a window in which proteins adsorb and coat urinary crystals (support of excretion) without affecting crystallization. Strongest effects on crystallization were observed for polypeptides that are either highly hydrophilic (\u3e950 kJ/mol) and highly carboxylated (poly asp, poly glu), or else highly hydrophilic and highly phosphorylated (native OPN isoforms), suggesting that highly hydrophilic proteins strongly affect precipitation processes in the urinary tract. Therefore, the level of hydrophilicity and net charge is a critical factor in the ability of polypeptides to affect crystallization and to regulate biomineralization processes