Covalent immobilization of the multienzyme enniatin synthetase

Summary The multienzyme enniatin synthetase was covalently immobilized to N-hydroxysuccinimide activated agarose. The stability of the immobilized enzyme at 25°C was enhanced compared to the soluble enzyme. Immobilization experiments also indicated that the enniatins are synthesized by a single molecule and thus do not require interactions of several enzyme molecules.     

Selective synthesis of depsipeptides by the immobilized multienzyme enniatin synthetase

Summary The multifunctional enzyme enniatin synthetase was immobilized by adsorption to propyl agarose. The immobilized multienzyme retained 45% of the activity of the free enzyme; an operational half-life of about 15 h was estimated. Selective synthesis of several different enniatin homologues was achieved with propyl agarose-bound enniatin synthetase. In addition to enniatin A, B, and C formation, a selective synthesis of non-naturally occurring depsipeptides, containing norvaline, norleucine, or -aminobutyric acid as sole amino acid moieties, was observed.     

Effect of Dietary Mono- and Polyunsaturated Fatty Acids on the Fatty Acid Composition of Pigs' Adipose Tissues

In two experiments with growing-finishing pigs six different dietary fats were added to a conventional diet (control - C) to study the effects of dietary monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) on the fatty acid composition of backfat and kidney fat at similar amounts of double bonds in feed (Exp. 1:7% pork fat - PF, 4.95% olive oil - OO, 3.17% soybean oil - SO) or a constant amount of 5% of processed fats (Exp. 2: partially hydrogenated fat - SAT, fractionated pork fats: olein - OLE, stearin - STE). Compared with the control, PUFA were only slightly increased in backfat of pigs fed PF, OLE, STE or OO, although dietary PUFA intake was up to 70% higher. With SO PUFA were significantly increased in adipose tissues, predominantly at the expense of MUFA. Consequently, a non-linear relationship was found between PUFA intake and proportion in backfat. MUFA were incorporated at the expense of SFA, therefore, adipose tissues of OO fed animals were lowest in SFA. Despite comparable amounts of double bonds in feed (Exp. 1), the degree of unsaturation measured as fat score (sum of double bonds) was in the order SO > OO > PF > C. In contrast, the proportion of SFA was C > PF = SO > OO. Regarding the decisive role of SFA for fat consistency it may be concluded that MUFA should also be considered in feeding recommendations for pigs. Furthermore, in case of a high dietary supply of MUFA, a simple index of double bonds might not be sufficiently conclusive to judge pig fat quality.     

Impact of mammalian cell culture conditions on monoclonal antibody charge heterogeneity: an accessory monitoring tool for process development

Recombinant monoclonal antibodies are predominantly produced in mammalian cell culture bioprocesses. Post-translational modifications affect the micro-heterogeneity of the product and thereby influence important quality attributes, such as stability, solubility, pharmacodynamics and pharmacokinetics. The analysis of the surface charge distribution of monoclonal antibodies provides aggregated information about these modifications. In this work, we established a direct injection pH gradient cation exchange chromatography method, which determines charge heterogeneity from cell culture supernatant without any purification steps. This tool was further applied to monitor processes that were performed under certain process conditions. Concretely, we were able to provide insights into charge variant formation during a fed-batch process of a Chinese hamster ovary cell culture, in turn producing a monoclonal antibody under varying temperatures and glucose feed strategies. Glucose concentration impacted the total emergence of acidic variants, whereas the variation of basic species was mainly dependent on process temperature. The formation rates of acidic species were described with a second-order reaction, where a temperature increase favored the conversion. This platform method will aid as a sophisticated optimization tool for mammalian cell culture processes. It provides a quality fingerprint for the produced mAb, which can be tested, compared to the desired target and confirmed early in the process chain.

     

Protein-free transfection of CHO host cells with an IgG-fusion protein: selection and characterization of stable high producers and comparison to conventionally transfected clones

In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development.     

A metal-coded affinity tag approach to quantitative proteomics

The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures.     

Genes: philosophical analyses put to the test

This paper describes one complete and one ongoing empirical study in which philosophical analyses of the concept of the gene were operationalized and tested against questionnaire data obtained from working biologists to determine whether and when biologists conceive genes in the ways suggested. These studies throw light on how different gene concepts contribute to biological research. Their aim is not to arrive at one or more correct 'definitions' of the gene, but rather to map out the variation in the gene concept and to explore its causes and its effects.     

Mistletoe viscotoxins induce membrane permeabilization and spore death in phytopathogenic fungi

Viscotoxins (Vts) are basic peptides expressed in mistletoe leaves, seeds and stems which have been shown to be cytotoxic to mammalian cells. The aim of this study was to analyse whether Vts were able to control and/or inhibit the growth of phytopathogenic fungi to obtain a clue to their biological function. Incubation of two Vt isoforms, VtA₃ and VtB, at a final concentration of 10 µ M resulted in a complete blockage of the germination of spores from three different pathogenic fungi. It was also shown that lower concentrations than 10 µ M of VtA₃ and VtB inhibit their mycelial growth in a dose-dependent manner. The protein dose required to inhibit the growth of Fusarium solani and Sclerotinia sclerotiorum to a 50% was between 1.5 and 3.75 µ M , which represents a potent activity. No significant differences in the antifungal potency for each Vt isoform, either VtA₃ and VtB, were observed, although they have been shown to exert differential cytotoxicity on mammalian cells. It was also demonstrated that Vts act as fungicidal compounds. To explore the basis of the antifungal activity the ability of VtA₃ to induce changes in membrane permeability and on the oxidative status of F. solani spores was analysed. By using a specific fluorescent probe on intact spores, it was demonstrated that VtA₃ produces rapid changes in fungal membrane permeability. It also induces H₂O₂ production verified by a histochemical staining. The data presented in this study support a direct role of Vts in the plant defence determined by their lethal effect on fungal pathogens.     

The crossflow injection technique: an improvement of the ethanol injection method

A novel scalable liposome preparation technique for pharmaceutical application is presented. Previous experiments have shown that the concept of continuous crossflow injection is a promising approach. For the characterization of the process, we focus on the influencing parameters like the lipid concentration, the injection hole diameter, the injection pressure, the buffer flow rate, and system performance. These experiments demonstrate that the injection hole diameter and the system performance do not influence the vesicle forming process and that a minimum of buffer flow rate is required to affect batch homogeneity. In contrast, strongly influencing parameters are lipid concentration in combination with increasing injection pressures. After exceeding the upper pressure limit of the linear range, where injection velocities remain constant, the vesicle batches are narrowly distributed, also when injecting higher lipid concentrations. Reproducibility and scalability data show similar results with respect to vesicle size and size distribution and demonstrate the stability and robustness of the novel continuous liposome preparation technique.