A Rapid Method for the Recovery,Quantification and Electrophoretic Analysis of Proteins from Beer

A previously‐developed method for protein recovery from wine has been applied to beer and beer foam samples. The method involves the complexation of proteins with dodecyl sulfate (added as sodium salts) and subsequently the insolubilization of the protein‐detergent complexes by addition of potassium ions (added as KCl). The protocol allows preparation of proteins from a few hundred microliters of beverage in a few minutes. The precipitated proteins are free from interfering materials and are directly utilizable for quantitative and electrophoretic assays.     

IpO: plasmids and methods for simplified,PCR‐based DNA transplant in yeast

Many yeast experiments require strains modified by recombinant DNA methods. Some experiments require precise insertion of a DNA segment into the genome without a selectable marker remaining. For these applications, we developed a new PCR‐based method for marker‐free DNA transplant. The current PCR‐based method requires the labour‐intensive construction of a PCR template plasmid with repeats of the DNA segment flanking URA3. The design of a new vector, IpO, reduces the work in cloning a single copy of the DNA segment between overlapping URA3 fragments present in the vector. Two PCRs are performed that capture the DNA segment and one or the other URA3 fragment. When the PCR products are co‐transformed into yeast, recombination between the overlapping URA3 fragments restores URA3 and transposes the cloned DNA segment inside out, creating a repeat‐URA3‐repeat cassette. Sequences designed into the PCR primers target integration of the cassette into the genome. Subsequent selection with 5‐fluoro‐orotic acid yields strains that have 'popped out' URA3 via recombination between the DNA repeats, with the result being the precise insertion of the DNA segment minus the selectable marker. An additional advantage of the IpO method is that it eliminates PCR artifacts that can plague the current method's repeat‐containing templates. Copyright © 2014 John Wiley & Sons, Ltd.     

叶蛋白提取分离及应用研究进展

叶蛋白因具有资源丰富、营养价值高、不含动物性胆固醇等特点而备受关注,是一种具有良好开发价值的新型蛋白资源。细胞破碎的方法主要有研磨、组织捣碎等方法;提取的方法主要有酸（碱）加热提法、发酵法、有机溶剂沉淀等方法;分离纯化的方法主要有电泳、色谱等方法。叶蛋白在医药、食品、饲料等方面都有广泛的应用。     

Revised procedures for yeast metabolites extraction: application to a glucose pulse to carbon-limited yeast cultures, which reveals a transient activation of the purine salvage pathway

In this study we have revised our original procedure of yeast metabolites extraction. We showed that: (a) less than 5% of intracellular metabolites leaks out during the step of rapid arrest of cellular metabolism by quenching yeast cells into a 60% methanol solution kept at -40 degrees C; and (b) with a few exception, the stability of metabolites were not altered during the 3 min boiling procedure in a buffered ethanol solution. However, there was a loss of external added metabolites of 5-30%, depending on the type of metabolites. This was mainly attributable to their retention on cellular debris after ethanol treatment, which prevented centrifugation of the cellular extracts before evaporation of ethanol. We further simplified our previous high-performance ionic chromatography (HPIC) techniques for easier, more reliable and robust quantitative measurements of organic acids, sugar phosphates and sugar nucleotides, and extended these techniques to purine and pyrimidine bases, using a variable wavelength detector set at 220 and 260 nm in tandem with a pulsed electrochemical or suppressed conductivity detector. These protocols were successfully applied to a glucose pulse to carbon-limited yeast cultures on purines metabolism. This study showed that glucose induced a fast activation of the purine salvage pathway, as indicated by a transient drop of ATP and ADP with a concomitant rise of IMP and inosine. This metabolic perturbation was accompanied by a rapid increase in the activity of the ISN1-encoded specific IMP-5'-nucleotidase. The mechanism of this activation remains to be determined.     

The MAGi RNA extraction method: a highly efficient and simple procedure for fresh and dry plant tissues

BACKGROUND: Samples from different plant species, different organs or tissues at different times of the year, usually show great differences in their cell compositions, pH, and the endogenous RNase activities, decreasing the RNA yield and quality. RESULTS: In this study we describe a reagent and a simple total RNA isolation method for plant organs, tissues and dry seeds. The RNA extraction reagent (MAGi) is non‐toxic and can be stored at room temperature for several months to years. The principle of the total RNA extraction is that tissues are lysed in extraction solution with the aid of mortar homogenization–maceration, and cellular proteins, polysaccharides and DNA are removed from the RNA. We tested the reported method on more than 16 different types of plant seed and 15 different tissues and organs of pepper. CONCLUSION: The RNA extraction procedure reported in the present study greatly reduces the time required to isolate dry seed total RNA and other tissues by more than half as compared with the previously reported methods. The range of typical RNA yield and quality represents a significant improvement over existing protocols. The quality is high enough to be considered as suitable method for RT‐PCR, cDNA library construction and microarray gene expression studies. Copyright © 2008 Society of Chemical Industry     

一种适用于油菜、大豆、花生、芝麻四种油料作物种子的RNA提取方法

油料作物种子中,大量油脂、蛋白、酚类物质的存在和易氧化的特点导致RNA提取困难。为了解决这一问题,本研究在商业化RNA提取试剂盒的基础上,通过以下3方面的改进,使之成功应用于油菜、花生、芝麻和大豆的各个时期种子RNA提取和后期的荧光定量分析实验：1）增加抗氧化剂巯基乙醇和PVP;2）根据不同作物的油脂含量调整氯仿的比例;3）改进RNA沉淀方法,加大洗脱液体积后用乙醇沉淀。与常规的CTAB、TRIZOL及文献报道的RNA提取方法比较,本方法提取的RNA更完整、得率更高、纯度更高;所得RNA被成功应用于FAD2基因的荧光定量表达分析,发现了四种作物种子不同发育时期的FAD2基因的表达差异,证明了改良方法的有效性。     

An in vivo detection system for transient and low‐abundant protein interactions and their kinetics in budding yeast

Methylation tracking (M‐Track) is a protein‐proximity assay in Saccharomyces cerevisiae, allowing the detection of transient protein–protein interactions in living cells. The bait protein is fused to a histone lysine methyl transferase and the prey protein to a methylation acceptor peptide derived from histone 3. Upon interaction, the histone 3 fragment is stably methylated on lysine 9 and can be detected by methylation‐specific antibodies. Since methylation marking is irreversible in budding yeast and only takes place in living cells, the occurrence of artifacts during cell lysate preparation is greatly reduced, leading to a more accurate representation of native interactions. So far, this method has been limited to highly abundant or overexpressed proteins. However, many proteins of interest are low‐abundant, and overexpression of proteins may interfere with their function, leading to an artificial situation. Here we report the generation of a toolbox including a novel cleavage‐enrichment system for the analysis of very low‐abundant proteins at their native expression levels. In addition, we developed a system for the parallel analysis of two prey proteins in a single cell, as well as an inducible methylation system. The inducible system allows precise control over the time during which the interaction is detected and can be used to determine interaction kinetics. Furthermore, we generated a set of constructs facilitating the cloning‐free genomic tagging of proteins at their endogenous locus by homologous recombination, and their expression from centromeric plasmids. GenBank submissions: pCK900; KM407502, pCK901; KM407503, pCK902; KM407504, pCK903; KM407505, pCK904; KM407506, pCK905; KM407507, pCK906; KM407508, pCK907; KM407509, pCK908; KM407510, pCK909; KM407511, pCK910; KM407512, pCK911; KM407513. © 2015 The Authors. Yeast published by John Wiley & Sons Ltd.     

Near-infrared spectroscopic monitoring of biomass, glucose, ethanol and protein content in a high cell density baker's yeast fed-batch bioprocess

The use of at-line NIRS to monitor a high cell density fed-batch baker's yeast bioprocess was investigated. Quantification of the key analytes (biomass, ethanol and glucose) and the product quality indicator (percentage protein content) was studied. Biomass was quantitatively modelled using whole matrix samples (as was percentage protein content). The dominance of the whole matrix spectrum by biomass, and its associated light scattering effects, were overcome by use of filtrate samples and adapted (semi-synthetic) filtrate samples, which allowed successful ethanol and glucose modelling, respectively. Calibrations were rigorously challenged via external validation with large sample sets relative to the calibration sample size, ensuring model robustness and potential practical utility. The standard errors of calibration for biomass, glucose, ethanol and total intracellular protein were (g/l) 1.79, 0.19, 0.79 and 0.91, respectively, comparable to those of the primary assays. The calibration strategies necessary to generate quantitative models for this range of analytes in such a complex high cell density bioprocess fluid are discussed.     

适合酿酒葡萄果实、果皮及种子的蛋白质提取方法

制备高质量的蛋白质样品是进行蛋白免疫印迹(Western blot)分析的前提.酿酒葡萄果实中含有大量的多酚和多糖等物质,给蛋白质的提取带来了因难.本研究比较了不同聚乙烯聚吡咯烷酮(PVPP)浓度和料液比对赤霞珠葡萄果实、果皮和种子中粗蛋白提取的影响.结果表明,采用10%PVPP及1:2料液比对果实、5%PVPP及1:3料液比对果皮或10%PVPP及1:3料液比对种子的蛋白进行提取,可获得较好的SDS-PAGE图谱;对花色素还原酶(ANR)的蛋白免疫印迹分析显示,ANR在果实、果皮和种子中均有表达,分子量约为43kDa.     

Sdo1p,the yeast orthologue of Shwachman–Bodian–Diamond syndrome protein,binds RNA and interacts with nuclear rRNA‐processing factors

The Shwachman–Bodian–Diamond syndrome protein (SBDS) is a member of a highly conserved protein family of not well understood function, with putative orthologues found in different organisms ranging from Archaea, yeast and plants to vertebrate animals. The yeast orthologue of SBDS, Sdo1p, has been previously identified in association with the 60S ribosomal subunit and is proposed to participate in ribosomal recycling. Here we show that Sdo1p interacts with nucleolar rRNA processing factors and ribosomal proteins, indicating that it might bind the pre‐60S complex and remain associated with it during processing and transport to the cytoplasm. Corroborating the protein interaction data, Sdo1p localizes to the nucleus and cytoplasm and co‐immunoprecipitates precursors of 60S and 40S subunits, as well as the mature rRNAs. Sdo1p binds RNA directly, suggesting that it may associate with the ribosomal subunits also through RNA interaction. Copyright © 2009 John Wiley & Sons, Ltd.     

Inactivation mechanisms of electron beam irradiation on Listeria innocua through the integrity of cell membrane,genomic DNA and protein structures

As a non-thermal sterilisation technology, electron beam irradiation (EBI) has attracted great interest for microbial inactivation in food preservation. This study aims to investigate the effects of EBI on membrane permeability, physiological status, morphological structure, genome integrity and protein structures of Listeria innocua irradiated at doses of 0.75, 1.50, 2.25, 3.00, 3.75 and 5.00 kGy. The results showed that EBI noticeably reduced the total microbial counts of L. innocua by more than 7 log CFU mL⁻¹ with 5.00 kGy treatment. The cell membrane permeability increased, resulting in the leakage of intracellular substances and changes in cell physiological status, which was proven by the cell staining and electron microscopy (EM) observations. Moreover, the integrity of genomic DNA and protein secondary structure, but not the protein primary structure were also disrupted. These findings provide the intrinsic mechanisms for the inactivation of L. innocua affected by EBI, which could serve as a theoretical basis for a better application of EBI in food sterilisation.     

Wine yeast molecular typing using a simplified method for simultaneously extracting mtDNA,nuclear DNA and virus dsRNA

Quick and accurate methods are required for the identification of industrial, environmental, and clinical yeast strains. We propose a rapid method for the simultaneous extraction of yeast mtDNA, nuclear DNA, and virus dsRNA. It is simpler, cheaper, and faster than the previously reported methods. It allows one to choose among a broad range of molecular analysis approaches for yeast typing, avoiding the need to use of several different methods for the separate extraction of each nucleic acid type. The application of this method followed by the combined analysis of mtDNA and dsRNA (ScV-M and W) is a highly attractive option for fast and efficient wine yeast typing.     

Free and immobilized Lactobacillus casei ATCC 393 on whey protein as starter cultures for probiotic Feta-type cheese production

The use of free and immobilized Lactobacillus casei ATCC 393 on whey protein as starter culture in probiotic Feta-type cheese production was evaluated. The probiotic cultures resulted in significantly higher acidity; lower pH; reduced counts of coliforms, enterobacteria, and staphylococci; and improved quality characteristics compared with cheese with no culture. Microbiological and strain-specific multiplex PCR analysis showed that both free and immobilized L. casei ATCC 393 were detected in the novel products at levels required for conferring a probiotic effect at the end of the ripening. The effect of starter culture on production of volatile compounds was investigated by the solid-phase microextraction gas chromatography-mass spectrometry analysis technique. The immobilized cells resulted in an improved profile of aroma-related compounds and the overall high quality of the novel products was ascertained by the preliminary sensory test. Finally, the high added value produced by exploitation of whey, which is an extremely polluting industrial waste, was highlighted and assessed.