Comparison of protein quantification and extraction methods suitable for E-coli cultures

Many different extraction and analysis methods exist to determine the protein fraction of microbial cells. For metabolic engineering purposes it is important to have precise and accurate measurements. Therefore six different protein extraction protocols and seven protein quantification methods were tested and compared. Comparison was based on the reliability of the methods and boxplots of the normalized residuals. Some extraction techniques (SDS/chloroform and toluene) should never be used: the measurements are neither precise nor accurate. Bugbuster extraction combined with UV280 quantification gives the best results, followed by the combinations sonication-UV280 and EasyLyse-UV280. However, if one does not want to use the quantification method UV280, one can opt to use Bugbuster, EasyLyse or sonication extraction combined with any quantification method with exception of the EasyLyse-BCA_P and sonication-BCA_P combinations

Production of protein extracts from Swedish red, green, and brown seaweeds, Porphyra umbilicalis Kützing, Ulva lactuca Linnaeus, and Saccharina latissima (Linnaeus) J. V. Lamouroux using three different methods

peer-reviewedThe demand for vegetable proteins increases globally and seaweeds are considered novel and promising protein sources. However, the tough polysaccharide-rich cell walls and the abundance of polyphenols reduce the extractability and digestibility of seaweed proteins. Therefore, food grade, scalable, and environmentally friendly protein extraction techniques are required. To date, little work has been carried out on developing such methods taking into consideration the structural differences between seaweed species. In this work, three different protein extraction methods were applied to three Swedish seaweeds (Porphyra umbilicalis, Ulva lactuca, and Saccharina latissima). These methods included (I) a traditional method using sonication in water and subsequent ammonium sulfate-induced protein precipitation, (II) the pH-shift protein extraction method using alkaline protein solubilization followed by isoelectric precipitation, and (III) the accelerated solvent extraction (ASE®) method where proteins are extracted after pre-removal of lipids and phlorotannins. The highest protein yields were achieved using the pH-shift method applied to P. umbilicalis (22.6 ± 7.3%) and S. latissima (25.1 ± 0.9%). The traditional method resulted in the greatest protein yield when applied to U. lactuca (19.6 ± 0.8%). However, the protein concentration in the produced extracts was highest for all three species using the pH-shift method (71.0 ± 3.7%, 51.2 ± 2.1%, and 40.7 ± 0.5% for P. umbilicalis, U. lactuca, and S. latissima, respectively). In addition, the pH-shift method was found to concentrate the fatty acids in U. lactuca and S. latissima by 2.2 and 1.6 times, respectively. The pH-shift method can therefore be considered a promising strategy for producing seaweed protein ingredients for use in food and feed

A realistic assessment of methods for extracting gene/protein interactions from free text

Background: The automated extraction of gene and/or protein interactions from the literature is one of the most important targets of biomedical text mining research. In this paper we present a realistic evaluation of gene/protein interaction mining relevant to potential non-specialist users. Hence we have specifically avoided methods that are complex to install or require reimplementation, and we coupled our chosen extraction methods with a state-of-the-art biomedical named entity tagger. Results: Our results show: that performance across different evaluation corpora is extremely variable; that the use of tagged (as opposed to gold standard) gene and protein names has a significant impact on performance, with a drop in F-score of over 20 percentage points being commonplace; and that a simple keyword-based benchmark algorithm when coupled with a named entity tagger outperforms two of the tools most widely used to extract gene/protein interactions. Conclusion: In terms of availability, ease of use and performance, the potential non-specialist user community interested in automatically extracting gene and/or protein interactions from free text is poorly served by current tools and systems. The public release of extraction tools that are easy to install and use, and that achieve state-of-art levels of performance should be treated as a high priority by the biomedical text mining community

Evaluation of a two-phase extraction system of carbohydrates and proteins from chlorella vulgaris utex 1803

Microalgae are a valuable source of high-value products and biofuels, however the high-energy cost required for the extraction of their metabolites has kept questioning on possible industrial upgrading. The aim of this study was to evaluate the effect of temperature, solvent/biomass, NaOH concentration and thermal pretreatment of the biomass in a 2-cycle carbohydrate and protein extraction system. Results shown that best conditions for carbohydrates extraction are achieved at a solvent concentration of 3.67 M, 55°C and a solvent/biomass ratio of 30mL/g. On the other side, the best conditions for protein were 3 M, 85°C and 45 mL/g. The efficiencies achieved under these conditions were 95% for carbohydrates and 98% for proteins. Using the best extraction conditions for each metabolite a thermal pre-treatment was performed at 25°C, 75°C and 105°C. Results indicate that highest efficiencies were achieved with dry biomass pretreated at 105°C, with values of 95% for carbohydrates and 98% for proteins

Microalgae cultivation for lipids and carbohydrates production

Microalgae are photoautotrophic microorganisms that can produce energy both by using sunlight, water and CO2 (phototrophic metabolism) and by using organic sources such as glucose (heterotrophic metabolism). Heterotrophic growth is a key factor in microalgae research, due to its increased productivity and the lower capital and operative costs compared to photoautotrophic growth in photobioreactors. Carbohydrate production from microalgae is usually investigated for the production of biofuels (e.g. bioethanol) by successive fermentation, but also other applications can be envisaged in biopolymers. In this work an increment in carbohydrate purity after lipid extraction was found. Protein hydrolysis for different microalgae strains (Scenedesmus sp. and Chlorella sp.) was investigated. Microalgae were cultivated under photoautotrophic or heterotrophic conditions, collecting biomass at the end of the growth. Biomass samples were dried or freeze dried and used for carbohydrate and lipid extraction tests. Lipid extraction was achieved using different organic solvents (methanol-chloroform and hexane-2propanol). Basic protein hydrolysis has been carried out testing different temperatures and NaOH concentrations values. Lipids were spectrophotometrically quantified, while residual biomass was saccharificated and the total amount of sugars was measured. Significant differences about the purity of extracted carbohydrates were found comparing dried with freeze dried biomass. However, not a very promising purification of carbohydrates was achieved after protein hydrolysis, asking for further analysis. © Copyright 2017, AIDIC Servizi S.r.l

Preparation of nuclear matrices from cultured cells: subfractionation of nuclei in situ

Analyses of the different structural systems of the nucleus and the proteins associated with them pose many problems. Because these systems are largely overlapping, in situ localization studies that preserve the in vivo location of proteins and cellular structures often are not satisfactory. In contrast, biochemical cell fractionation may provide artifactual results due to cross-contamination of extracts and structures. To overcome these problems, we have developed a method that combines biochemical cell fractionation and in situ localization and leads to the preparation of a residual cellular skeleton (nuclear matrix and cytoskeletal elements) from cultured cells. This method's main feature is that cell fractionation is performed in situ. Therefore, structures not solubilized in a particular extraction step remain attached to the substrate and retain their morphology. Before and after each extraction step they can be analyzed for the presence and location of the protein under study by using immunological or cytochemical techniques. Thereby the in vivo origin of a protein solubilized in a particular extraction step is determined. The solubilized protein then may be further characterized biochemically. In addition, to allow analyses of proteins associated with the residual cellular skeleton, we have developed conditions for its solubilization that do not interfere with enzymatic and immunological studies