A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures

<p>Abstract</p> <p>Background</p> <p>Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in <it>Escherichia coli </it>in simple shaken cultures.</p> <p>Results</p> <p>The enzymatic glucose release system together with a well-balanced combination of mineral salts and complex medium additives provided high cell densities, high protein yields and a considerably improved proportion of soluble proteins in harvested cells. The cultivation method consists of three steps: 1) controlled growth by glucose-limited fed-batch to OD₆₀₀~10, 2) addition of growth boosters together with an inducer providing efficient protein synthesis within a 3 to 6 hours period, and 3) a slow growth period (16 to 21 hours) during which the recombinant protein is slowly synthesized and folded. Cell densities corresponding to 10 to 15 g l^-1cell dry weight could be achieved with the developed technique. In comparison to standard cultures in LB, Terrific Broth and mineral salt medium, we typically achieved over 10-fold higher volumetric yields of soluble recombinant proteins.</p> <p>Conclusions</p> <p>We have demonstrated that by applying the novel EnBase^®Flo cultivation system in shaken cultures high cell densities can be obtained without impairing the productivity per cell. Especially the yield of soluble (correctly folded) proteins was significantly improved in comparison to commonly used LB, Terrific Broth or mineral salt media. This improvement is thought to result from a well controlled physiological state during the whole process. The higher volumetric yields enable the use of lower culture volumes and can thus significantly reduce the amount of time and effort needed for downstream processing or process optimization. We claim that the new cultivation system is widely applicable and, as it is very simple to apply, could widely replace standard shake flask approaches.</p

Improved production of human type II procollagen in the yeast Pichia pastoris in shake flasks by a wireless-controlled fed-batch system

<p>Abstract</p> <p>Background</p> <p>Here we describe a new technical solution for optimization of <it>Pichia pastoris </it>shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in <it>P. pastoris </it>is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in <it>P. pastoris </it>is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control.</p> <p>Results</p> <p>By applying on-line pO₂monitoring we demonstrate that the widely used pulse feeding of methanol results in long phases of methanol exhaustion and consequently low expression of AOX1 controlled genes. Furthermore, we provide a solution to apply the fed-batch strategy in shake flasks. The presented solution applies a wireless feeding unit which can be flexibly positioned and allows the use of computer-controlled feeding profiles.</p> <p>By using the human collagen II as an example we show that a quasi-continuous feeding profile, being the simplest way of a fed-batch fermentation, results in a higher production level of human collagen II. Moreover, the product has a higher proteolytic stability compared to control cultures due to the increased expression of human collagen prolyl 4-hydroxylase as monitored by mRNA and protein levels.</p> <p>Conclusion</p> <p>The recommended standard protocol for methanol addition in shake flasks using pulse feeding is non-optimal and leads to repeated long phases of methanol starvation. The problem can be solved by applying the fed-batch technology. The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel. By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems.</p

Clinical and virological characteristics of hospitalised COVID-19 patients in a German tertiary care centre during the first wave of the SARS-CoV-2 pandemic: a prospective observational study

Purpose: Adequate patient allocation is pivotal for optimal resource management in strained healthcare systems, and requires detailed knowledge of clinical and virological disease trajectories. The purpose of this work was to identify risk factors associated with need for invasive mechanical ventilation (IMV), to analyse viral kinetics in patients with and without IMV and to provide a comprehensive description of clinical course. Methods: A cohort of 168 hospitalised adult COVID-19 patients enrolled in a prospective observational study at a large European tertiary care centre was analysed. Results: Forty-four per cent (71/161) of patients required invasive mechanical ventilation (IMV). Shorter duration of symptoms before admission (aOR 1.22 per day less, 95% CI 1.10-1.37, p < 0.01) and history of hypertension (aOR 5.55, 95% CI 2.00-16.82, p < 0.01) were associated with need for IMV. Patients on IMV had higher maximal concentrations, slower decline rates, and longer shedding of SARS-CoV-2 than non-IMV patients (33 days, IQR 26-46.75, vs 18 days, IQR 16-46.75, respectively, p < 0.01). Median duration of hospitalisation was 9 days (IQR 6-15.5) for non-IMV and 49.5 days (IQR 36.8-82.5) for IMV patients. Conclusions: Our results indicate a short duration of symptoms before admission as a risk factor for severe disease that merits further investigation and different viral load kinetics in severely affected patients. Median duration of hospitalisation of IMV patients was longer than described for acute respiratory distress syndrome unrelated to COVID-19

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Erstellen von künstlichen Zuckerisomerasen basierend auf dem Proteingerüst einer monomeren Triosephosphat-Isomerase (A-TIM) mittels Protein-Engineering

Biokatalysatoren werden heutzutage verbreitet in einer Vielfalt von industriellen Prozessen eingesetzt. Die meisten Biokatalysatoren sind jedoch nicht an die extremen Bedingungen in industriellen Prozessen angepasst. Deshalb, nutzt das sogenannte Protein-Engineering die Techniken der Molekularbiologie, um maßgeschneiderte künstliche Biokatalysatoren zu entwerfen, die für Anwendung in der Industrie optimiert sind. Von besonderem Interesse für die Industrie ist hier die Produktion von chiralen Molekülen, z.B. a-Hydroxyaldehyden wie Monosacchariden, die sich daher als mögliche Ausgangspunkte eignen. Enzyme, die Monosaccharide umsetzen eignen sich sehr gut für einen Protein-Engineering-Ansatz. Das Projektziel der vorliegenden Arbeit war der Transfer einer in der Natur vorkommenden Enzymaktivität auf eine bekannte, häufig vorkommende Proteinstruktur. Genauer gesagt, sollte die Aktivität von drei verschiedenen Zuckerisomerasen (D-Ribose-5-Phosphat Isomerase A, DXylose Isomerase A, L-Arabinose Isomerase A) je auf die TIM-Fassstruktur einer monomeren Variante der Triosephosphat-Isomerase (A-TIM) übertragen werden. Wir vermuten, dass sich die Substratspezifität sehr leicht verändern lässt, da die TIM-Fassstruktur eine große Vielfalt in ihrer Funktionalität aufweist. Dafür wurde ein passendes in vivo Selektionssystem entwickelt, einschliesslich spezifischer E.coli Knockoutstämme. 16 verschiedene Genbibliotheken des Zielgens (A-TIM) wurden mittels eines gerichteten Evolutionsansatzes und eines strukturbasiertem rationalen Design-Ansatzes. Es wurden einige positive Klone mit L-arabinose Isomerase Aktivität gefunden. Jedoch keine für andere Aktivitäten. Diese Varianten wurden durch Sequenzieren, biochemische und –physikalische sowie anschließende Proteinkristallisation analysiert. Mutationen fanden sich hauptsächlich am C-terminalen Ende der TIM-Fassstruktur, welches dem katalytischen Ende des Enzyms entspricht, befinden, nicht aber am N-terminale Ende der TIM-Fassstruktur, welches dem Stabilitätsende des Enzyms entspricht. Die Sekundärstrukturveränderungen waren minimal und der vermeintliche Reaktionsmechanism scheint gleich dem von TIM zu sein, mit einem Glutamat, welches als Base fungiert. Die katalytische Aktivität einer Isomerase konnte erfolgreich auf eine andere Isomerase übertragen werden. Dies geschah unter Verlust der Kofaktor-Abhängigkeit.Biocatalysts are nowadays commonly applied in many industrial production processes. Most biocatalysts however, are not adapted to the extreme conditions present in industrial processes. Therefore, protein engineering makes use of the tools available in molecular biology to design tailor-made non-natural biocatalysts optimized for the application in industry. Of special industrial interest is the production of chiral molecules, e.g. a-hydroxyaldehydes such as monosaccharides which serve as a feasible starting point. Enzymes converting them can be targeted easily by protein engineering. This work aimed to transfer an existing enzyme activity onto a known common enzyme fold. Specifically, the activity of three different sugar isomerases (D-ribose-5-phosphate isomerase A, D-xylose isomerase A, L-arabinose isomerase A) was to be transplanted onto the TIM-barrel scaffold of a monomeric triosephosphate isomerase (A-TIM). We hypothesize that that since this protein fold exhibits vast diversity in functionality in nature it could be rather straightforward to change the substrate specificity. For this a suitable in vivo selection system was developed including specific E.coli knockout strains. 16 different gene libraries of the target gene (A-TIM) were created via a directed evolution and a structure-based rational design approach. Several positive hits were found displaying L-arabinose isomerase activity; not for any other activity though. The found variants were analyzed by sequencing, biochemical and biophysical methods and finally protein crystallography. Mutations of found variants were mainly found at the C-terminal end of the TIM-barrel which corresponds to the catalytic end of the enzyme while the N-terminal end responsible for the protein stability did not show mutations. It was found that the secondary structure changes were minimal and the putative reaction mechanism would be the same as for TIM using the E167 as the critical base. The catalytic activity was successfully transferred from one isomerase to another while the dependency on any cofactors was lost

Exosomes as renal inductive signals in health and disease, and their application as diagnostic markers and therapeutic agents

Cells secrete around 30-1000 nm membrane-enclosed vesicles, of which members of the subgroup between 30-100 nm are termed exosomes (EXs). EXs are released into the extracellular space and are widely present in body fluids and incorporated mRNA, miRNA, proteins and signaling molecules. Increasing amounts of evidence suggest that EXs play an important role not only in cell-to-cell communication but also in various physiological and disease processes. EXs secreted by kidney cells control nephron function and are involved in kidney diseases and cancers. This makes them potential targets for diagnostic and therapeutic applications such as non-invasive biomarkers and cell-free vaccines and for use as drug delivery vehicles. This review provides an overview on the known roles of EXs in kidney development and diseases, including renal cancer. Additionally, it covers recent findings on their significance as diagnostic markers and on therapeutic applications to renal diseases and cancers. The intention is to promote an awareness of how many questions still remain open but are certainly worth investigating

Wnt Signaling in Renal Cell Carcinoma

Renal cell carcinoma (RCC) accounts for 90% of all kidney cancers. Due to poor diagnosis, high resistance to the systemic therapies and the fact that most RCC cases occur sporadically, current research switched its focus on studying the molecular mechanisms underlying RCC. The aim is the discovery of new effective and less toxic anti-cancer drugs and novel diagnostic markers. Besides the PI3K/Akt/mTOR, HGF/Met and VHL/hypoxia cellular signaling pathways, the involvement of the Wnt/β-catenin pathway in RCC is commonly studied. Wnt signaling and its targeted genes are known to actively participate in different biological processes during embryonic development and renal cancer. Recently, studies have shown that targeting this pathway by alternating/inhibiting its intracellular signal transduction can reduce cancer cells viability and inhibit their growth. The targets and drugs identified show promising potential to serve as novel RCC therapeutics and prognostic markers. This review aims to summarize the current status quo regarding recent research on RCC focusing on the involvement of the Wnt/β-catenin pathway and how its understanding could facilitate the identification of potential therapeutic targets, new drugs and diagnostic biomarkers