Recent advances and challenges in the recovery and purification of cellular exosomes

Exosomes are nanovesicles secreted by most cellular types that carry important biochemical compounds throughout the body with different purposes, playing a preponderant role in cellular communication. Because of their structure, physicochemical properties and stability, recent studies are focusing in their use as nanocarriers for different therapeutic compounds for the treatment of different diseases ranging from cancer to Parkinson's disease. However, current bioseparation protocols and methodologies are selected based on the final exosome application or intended use and present both advantages and disadvantages when compared among them. In this context, this review aims to present the most important technologies available for exosome isolation while discussing their advantages and disadvantages and the possibilities of being combined with other strategies. This is critical since the development of novel exosome‐based therapeutic strategies will be constrained to the effectiveness and yield of the selected downstream purification methodologies for which a thorough understanding of the available technological resources is needed.     

A highly active fungal β-glucosidase

A highly active thermostable β-glucosidase was purified to homogeneity from a strain ofTrichoderma sp. The enzyme was an extracellular glycoprotein and showed hydrolytic activity toward several β-glucosides. Cellobiose was found to be the substrate of choice for this enzyme. This finding could suggest future technological applications of the purified protein.     

Taura 综合征病毒主要结构蛋白的表达与纯化

根据Taura综合征病毒(TSV)基因组,设计特异性引物,从感染病毒组织中提取组织总RNA后扩增,分别将3个主要结构蛋白基因VP1、VP2和VP3克隆到pGEM TEasyVector.与表达载体连接后,导入大肠杆菌中诱导表达,并纯化目的蛋白.诱导表达的融合蛋白分子量分别为54.2×103、43×103和57.1×103,在变性条件下过柱纯化VP1和VP2,一次可以纯化10mg以上纯度较高的蛋白.     

Reversible 5′-end biotinylation and affinity purification of synthetic RNA

A reversible biotinylation phosphoramidite was synthesized and incorporated onto the 5′-end of an oligoribonucleotide on a solid phase synthesizer. After cleavage and deprotection, the crude synthetic oligomer mixture was incubated with NeutrAvidin^® coated microspheres, and the failure sequences removed by washing with a buffer followed by treating the microspheres with tetrabutylammonium fluoride to give a high quality unmodified full-length oligoribonucleotide.     

陆生植物黑麦草(Lolium multiflorum)对富营养化水体修复的围隔实验研究——氨氮的净化效应及其动态过程

采用浮床黑麦草（Lolium multi florum）对畜禽养殖富营养水体氨氮的净化效应及动态过程进行了研究．结果表明：浮床黑麦草对富营养水体中的氨氮具有明显的净化效果．当覆盖率30％时，对氨氮的去除率最高可达到95．89％．对其去除水体氨氮的动态过程拟合表明符合三次方程曲线．该研究为冬季陆生植物修复富营养水体提供重要依据．     

Genetic engineering to enhance the selectivity of protein separations

The ability to recover and purify natural and recombinant proteins, and the costs of doing so remain a major task in introducing the potential products of biotechnology. The bases for separation range from specific binding onto tailored reagents to solubility and partitioning behavior governed by a mixed bag of size, charge, and hydrophobicity. In most cases, a combination of methods is used in sequence, and improvements in the selectivity at an early stage can enhance the effectiveness of subsequent (and usually more costly) steps. Genetic engineering provides a means of improving the selectivity within the context of existing separation methods. By this strategy, improvements in selectivity are sought by bestowing a distinctive property on the protein of interest. The primary sequence of amino acids is altered, such that the protein can be selectively removed from other components of the multicomponent mixture in which such products are commonly found. In this article, the range of these “distinctive properties” and their pairing with various separation methods will be reviewed. Specific examples from our work, in which a distinctive charge is provided via a polypeptide “purification” fusion tail, will be discussed. Separation methods we have used with these fusion proteins are precipitation, two-phase aqueous extraction, reversed micellar extraction, and ion exchange using both resins and membranes.     

The high-temperature durability of Rh-free threeway catalysts used for automoble emission control

The high-temperature durability of cordierite honeycomb Rh-free three-way catalysts (TWC) containing additives has been studied by calcining catalysts at various temperatures in the interval of 600≈1100°C. The three-way performances of these catalysts were evaluated for the reaction of carbon monoxide(CO), hydrocarbon(HC) and nitrogen oxides(NOx) at given conditions by using a fixed bed reactor. The results show that after Rh-free TWC are thermally aged in air at 1100°C for 5h and 950°C for 104 h, the TWC still have better three-way activity compared with Rh-containing TWC.     

单分散交联聚甲基丙烯酸环氧丙酯树脂的制备及亲合纯化伴刀豆球蛋白-A

采用一步种子溶胀聚合法制备了颗粒呈单分散的交联聚甲基丙烯酸环氧丙南树脂，对其环氧基的组成比进行了表征，并以氨基葡萄糖为配基，首次制备了纯化粗品伴刀豆球蛋白-A(Ⅲ）(Con-A，Ⅲ)的聚合物基质的高效亲合色谱柱。配基在树脂上的键合量为8．2mg／g，对纯化后的Con-A（Ⅲ）的吸附量为13．4mg／g。使用该亲合色谱介质成功地从粗品Con-A中快速纯化了Con-A，电泳分析显示为一个主要的谱带，纯度从15％提高到95％。     

Characterization and separation of Al13 species using gel-filtration chromatography

A polyaluminum chloride (PAC) sample was prepared using a slow alkaline titration method. The Bio-Gel P-100 gel column chromatographic technique was used to separate and characterize the various forms of aluminum present in the prepared PAC solution. The effluents from a gel column were monitored using online chemical method: Al-Ferron timed complexation spectrophotometry and by ²⁷Al nuclear magnetic resonance (NMR) spectroscopy. Effects of different experimental conditions such as eluent flow rate, ionic strength and pH on separation of Al₁₃ were investigated. Experimental results indicated that molecule size exclusion was not the only parameter affecting the column chromatographic separation efficiency of Al₁₃ but molecule charge as well. Reducing the eluent flow rate, increasing the ionic strength and suitable pH resulted in increase in the separation efficiency. Experimental results clearly indicated that by varying the experimental conditions, it is possible to produce pure Al₁₃ species using a gel column chromatographic technique.     

Purification and partial characterization ofRhizomucor miehei lipase for ester synthesis

A commercialRhizomucor miehei lipase was purified by ammonium sulfate precipitation. Phenyl Sepharose 6 Fast Row hydrophobic interaction chromatography, and DEAE Sepharose Fast Flow anion-exchange chromatography. The recovery of lipase activity was 32% with a 42-fold purification. The molecular size of the purified enzyme was 31,600 Dalton and the pI 3.8. The enzyme was stable for at least 24 h within a pH range of 7.0-10.0, and 96.8% of the enzyme activity remained when kept at 30‡C for 24 h. Further, about 10–30% of the lipase activity was inhibited by K⁺, Li⁺, Ni⁺, Co²⁺, Zn²⁺, Mg²⁺, Sn²⁺, Cu²⁺, Ba²⁺, Ca²⁺, and Fe²⁺ ions and by SDS, but EDTA had no effect. Under the experimental conditions, the optimum temperature for the hydrolysis of olive oil was 50‡C (pH 8.0), and for the synthesis of 1-butyl oleate, 37‡C. It was concluded that hydrolytic activity of lipase alone is not a sufficient criterion for its synthetic potential. The optimal molar ratio of oleic acid and 1-butanol was 2:1 for 1-butyl oleate synthesis. The 1-butyl oleate yield was unaffected by purification of the enzyme after 12 h.