Progress towards continuous aqueous two-phase extraction via TAPPIR

At ICB II, we presented Aqueous Two-Phase Extraction (ATPE) as a non-chromatographic alternative for protein purification. We had developed an aqueous two-phase system with inexpensive and biocompatible PEG 1500 or 4000 and ammonium citrate. We purified several enzymes, more specifically a series of dehydrogenases [1], to near homogeneity after forward extraction into a PEG-heavy top phase at pH \u3e 9 and back extraction into a bottom phase at pH 4-6; in selected cases, we were able to obtain pure protein in the bottom phase without forward extraction into the top phase. Scale-up of the PEG 1500/4000-ammonium citrate to 5-10 L scale still often gave phase separation times of less than five minutes.[2] However, ATPE technology is characterized by complex phase separation and very limited number of separation stages not offering enough separation efficiency. Furthermore, conventional ATPE does not lend itself to continuous operation. Please click Additional Files below to see the full abstract

Neural response in obsessive-compulsive washers depends on individual fit of triggers

BackgroundPatients with obsessive-compulsive disorder (OCD) have highly idiosyncratic triggers. To fully understand which role this idiosyncrasy plays in the neurobiological mechanisms behind OCD, it is necessary to elucidate the impact of individualization regarding the applied investigation methods.This functional magnetic resonance imaging (fMRI) study explores the neural correlates of contamination/washing-related OCD with a highly individualized symptom provocation paradigm. Additionally, it is the first study to directly compare individualized and standardized symptom provocation. MethodsNineteen patients with washing compulsions created individual OCD hierarchies, which later served as instructions to photograph their own individualized stimulus sets. The patients and 19 case-by-case matched healthy controls participated in a symptom provocation fMRI experiment with individualized and standardized stimulus sets created for each patient. ResultsOCD patients compared to healthy controls displayed stronger activation in the basal ganglia (nucleus accumbens, nucleus caudatus, pallidum) for individualized symptom provocation. Using standardized symptom provocation, this group comparison led to stronger activation in the nucleus caudatus. The direct comparison of between-group effects for both symptom provocation approaches revealed stronger activation of the orbitofronto-striatal network for individualized symptom provocation.ConclusionsThe present study provides insight into the differential impact of individualized and standardized symptom provocation on the orbitofronto-striatal network of OCD washers. Behavioral and neural responses imply a higher symptom-specificity of individualized symptom provocation

Mass transfer of proteins in aqueous two-phase systems

Aqueous Two-Phase Extraction is known to be a gentle separation technique for biochemical molecules where product partitioning is fast. However, the reason for the high mass transfer rates has not been investigated, yet. Many researchers claim that the low interfacial tension facilitates the formation of very small droplets and with it a large interfacial area causing a fast partitioning. However, an experimental evidence for this hypothesis has not been published yet. In this study, the mass transfer coefficients of two proteins, namely lysozyme and bromelain, were determined by providing a defined interfacial area for partitioning. Compared to low molecular weight solutes the mass transfer coefficient for the proteins investigated was small proving for the first time that the large interfacial area and not fast diffusion seems to be the reason for fast protein partitioning

Molecular Interaction of Amino Acids with Acidic Zeolite BEA: The Effect of Water

Experimentally, zeolites were shown to adsorb amino acids from the aqueous phase. However, there has not been a satisfactory theoretical explanation on the effect of water on the underlying adsorption mechanisms yet. In this study, the effect of water on the pH-dependent adsorption behavior of glycine and alanine in zeolite BEA was investigated using density functional theory (DFT). Using a microsolvation model, the coadsorption and formation of an intermolecular H-bond system between water molecules, amino acid, and zeolite was shown. In addition, different pH values were modeled by varying the amino acids protonation states with the protonated states being significantly more stable in the BEA pores than the net neutral ones. This behavior was experimentally approved by isothermal titration calorimetry (ITC) and adsorption isotherm measurements. These results provide new molecular level information on the adsorption of amino acids in zeolites from the aqueous phase and could be used to support the experimental side of adsorption process design in industrial biotechnology by qualitatively predicting binding behavior by means of DFT calculations and simplified model systems

Selection and Use of Poly Ethylene Glycol and Phosphate Based Aqueous Two-Phase Systems for the Separation of Proteins by Centrifugal Partition Chromatography

<div><p>For an efficient separation with centrifugal partition chromatography (CPC) the selection of suitable biphasic phase systems is of crucial importance. Commonly, aqueous-organic systems have been widely used for the separation of natural products. Thus, a lot of knowledge about the selection of suitable systems and appropriate operation conditions of CPC is available. Alternatively, aqueous two-phase systems (ATPS) can be used in CPC. These systems offer the possibility to purify biotechnological substances like proteins. However, the selection and use of ATPS in CPC and accordingly the selection of appropriate operation conditions have rarely been investigated. In order to improve the knowledge, the focus of this research is on the selection of suitable ATPS and operation conditions for an efficient separation of biomolecules in CPC. Several systems, based on polyethylene glycol and phosphate salts have found to be suitable for use in CPC. Thereby, the hydrodynamic in the chamber is of major importance because it determines the stability and the performance during the separation run. An additional important aspect within the ATPS selection is the possibility to adjust the partition coefficient of the target compounds. This was achieved by changing the pH value of the system. Both aspects, hydrodynamic and adjustment of the partition coefficients were investigated in this study and the results obtained will help to select suitable ATPS and operating conditions for CPC separation of biomolecules.</p></div

Amino-Acid Adsorption in MFI-Type Zeolites Enabled by the pH-Dependent Ability to Displace Water

Downstream processing of biochemical products strongly depends on the interaction between biomolecules and material surfaces that can be influenced greatly by the pH level. In this study, the influence of the pH value on the adsorption of the amino acids glycine, alanine, and lysine in MFI-type zeolite was investigated using density functional theory (DFT) and microcalorimetric experiments. Different pH values were modeled by varying the amino acids protonation states. The investigated protonation states exhibit two different adsorption motifs in the pores, with the neutral α-C-amino group motifs being significantly less stable than the protonated ones. In the case of neutral glycine and alanine, the adsorption energy is insufficient to overcome the adsorption of the four water molecules usually present in the pores. This result explains the pH-dependent adsorption behavior that has also been observed experimentally and provides an avenue for designing efficient adsorption processes

Rrp5p, Noc1p and Noc2p form a protein module which is part of early large ribosomal subunit precursors in S. cerevisiae

Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p-Noc1p-Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity