Isolation of High-Purity Extracellular Vesicles by Extracting Proteins Using Aqueous Two-Phase System

We present a simple and rapid method to isolate extracellular vesicles (EVs) by using a polyethylene glycol/dextran aqueous two-phase system (ATPS). This system isolated more than ~75% of melanoma-derived EVs from a mixture of EVs and serum proteins. To increase the purity of EVs, a batch procedure was combined as additional steps to remove protein contaminants, and removed more than ~95% of the protein contaminants. We also performed RT-PCR and western blotting to verify the diagnostic applicability of the isolated EVs, and detected mRNA derived from melanoma cells and CD81 in isolated EVs.     

Separation of Flavonoids from Bamboo Leaves by Aqueous Two-Phase Extraction

以竹叶黄酮水提溶液为原料,采用PEG(聚乙二醇)/(NH4 )2SO4双水相体系对竹叶黄酮进行萃取,考察了PEG平均相对分子质量、PEG质量分数、(NH4)2SO4质量分数、pH值、NaCl质量分数、原液质量分数、萃取温度等对双水相及竹叶黄酮萃取效果的影响.双水相萃取法提取竹叶黄酮的最优条件为:PEG 400 31％,(NH4)2 SO4 11％,pH3.9,NaCl 0.7％,原液51.5％,萃取温度20℃,在此条件下得到的竹叶黄酮萃取率为97.8％.结果说明,双水相萃取法操作简单方便,成本低,不会引起生物质失活或变性,适合于黄酮类化合物的萃取分离.     

双水相萃取法分离竹叶黄酮的工艺研究

以竹叶黄酮水提溶液为原料,采用PEG(聚乙二醇)/(NH4)2SO4双水相体系对竹叶黄酮进行萃取,考察了PEG平均相对分子质量、PEG质量分数、(NH4)2SO4质量分数、pH值、NaCl质量分数、原液质量分数、萃取温度等对双水相及竹叶黄酮萃取效果的影响。双水相萃取法提取竹叶黄酮的最优条件为:PEG 400 31%,(NH4)2SO411%,pH 3.9,NaCl 0.7%,原液51.5%,萃取温度20℃,在此条件下得到的竹叶黄酮萃取率为97.8%。结果说明,双水相萃取法操作简单方便,成本低,不会引起生物质失活或变性,适合于黄酮类化合物的萃取分离。     

Rapid Method for the Isolation of Plasma Membranes from a Murine Fibrosarcoma Using an Aqueous Two-Phase Polymer System

An aqueous two-phase polymer system was used to isolate plasma membranes from a palpable mouse fibrosarcoma. The excised tumor tissue was washed with sterile saline and pushed through nylon screens of decreasing mesh size. This cell suspension was placed in Tris-buffered, isotonic sucrose plus MgSo₄ and homogenized by nitrogen cavitation. A pellet was collected from the homogenate by low-speed centrifugation and was added to the aqueous two-phase polymer system. After several brief, low-speed centrifugations, the interfacial material between the polymer phases was collected. Data from enzyme and biochemical assays demonstrated that this fraction was plasma membrane. This method provided a high yield of the surface membrane in less than three hours.     

植物蛋白质SUMO化修饰体外高效检测系统

蛋白质SUMO化修饰是一种调控蛋白命运的关键修饰方式, 广泛参与植物生长发育及逆境胁迫响应。SUMO化修饰过程主要由激活酶(E1)-结合酶(E2)-连接酶(E3)组成的级联酶促反应催化, 其关键酶组分将SUMO分子缀合至底物蛋白的赖氨酸残基, 形成共价异肽键以完成SUMO化修饰过程。该文报道了1种植物蛋白质SUMO化修饰体外高效检测系统, 通过在大肠杆菌(Escherichia coli)中构建拟南芥(Arabidopsis thaliana) SUMO化修饰的关键通路实现对底物蛋白的SUMO化修饰, 结果可通过免疫印迹进行检测。该系统可以简化植物蛋白质SUMO化修饰的检测流程, 为植物细胞SUMO化修饰的功能研究提供了有力工具。     

乙醇-磷酸氢二钾双水相体系萃取L-精氨酸

采用乙醇-磷酸氢二钾(K2HPO4)双水相体系萃取L-精氨酸。实验考察了乙醇浓度、K2HPO4浓度、pH、萃取温度对萃取分离L-精氨酸的影响。结果表明,L-精氨酸在该双水相体系的分配系数K随体系乙醇浓度、K2HPO4浓度的增大、萃取温度的升高而增大,随着体系pH的增大而减小;L-精氨酸在该双水相体系的萃取率随体系乙醇浓度和pH的增大而减小,随着体系K2HPO4浓度增大、萃取温度的升高而增大。     

Purification and Characterization of Polyphenol Oxidase From Waste Potato Peel by Aqueous Two-Phase Extraction

Potato peel from food industrial waste is a good source of polyphenol oxidase (PPO). This work illustrates the application of an aqueous two-phase system (ATPS) for the extraction and purification of PPO from potato peel. ATPS was composed of polyethylene glycol (PEG) and potassium phosphate buffer. Effect of different process parameters, namely, PEG, potassium phosphate buffer, NaCl concentration, and pH of the system, on partition coefficient, purification factor, and yield of PPO enzyme were evaluated. Response surface methodology (RSM) was utilized as a statistical tool for the optimization of ATPS. Optimized experimental conditions were found to be PEG1500 17.62% (w/w), potassium phosphate buffer 15.11% (w/w), and NaCl 2.08 mM at pH 7. At optimized condition, maximum partition coefficient, purification factor, and yield were found to be 3.7, 4.5, and 77.8%, respectively. After partial purification of PPO from ATPS, further purification was done by gel chromatography where its purity was increased up to 12.6-fold. The purified PPO enzyme was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by K_m value 3.3 mM, and V_max value 3333 U/mL, and enzyme stable ranges for temperature and pH of PPO were determined. These results revealed that ATPS would be an attractive option for obtaining purified PPO from waste potato peel.     

丙醇-硫酸铵双水相体系及应用探讨

通过研究丙醇-水-硫酸铵双水相分相情况,确定了刚好分相时丙醇和硫酸铵含量.测定了上、下相电导率和折光率,得出一定丙醇和硫酸铵含量分相后上、下相组成,上相主要富含丙醇,下相主要富含硫酸铵.将该双水相体系应用于西红柿和柠檬皮中色素分离,得到较好分离效果.     

An Inducible System for Rapid Degradation of Specific Cellular Proteins Using Proteasome Adaptors

A common way to study protein function is to deplete the protein of interest from cells and observe the response. Traditional methods involve disrupting gene expression but these techniques are only effective against newly synthesized proteins and leave previously existing and stable proteins untouched. Here, we introduce a technique that induces the rapid degradation of specific proteins in mammalian cells by shuttling the proteins to the proteasome for degradation in a ubiquitin-independent manner. We present two implementations of the system in human culture cells that can be used individually to control protein concentration. Our study presents a simple, robust, and flexible technology platform for manipulating intracellular protein levels.     

The Aqueous Two-Phase System Polyethylene Glycol/Dextran as a Reaction Medium for the Microsomal Monooxygenase System

An aqueous two-phase system of dextran and polyethylene glycol was investigated as a reaction medium for pig liver microsomes in order to find out if the partition of the microsomes, of the substrate 7-ethoxycoumarin and of the product 7-hydroxycoumarin favoured any biotechnological perspectives. Cytochrome-P-450 and NADPH-cytochrome P-450 reductase concentrations and the monooxygenase 7-ethoxycoumarin deethylation activity were measured under a variety of the system parameters. Microsomes totally partition into the bottom phase whereas the concentration ratio of substrate to product is higher in the microsome free top phase. An unfavourable effect is the specific partial deactivation of the cytochrome P-450 by polyethylene glycol.     

双水相体系萃取人参根中人参皂苷的研究

建立稳定的聚乙二醇(PEG)与(NH4)2SO4双水相体系以分离人参根中人参皂苷。通过上下相体积比(R)、分配系数(K)和回收率(Y)分析双水相体系对人参皂苷的萃取效果,研究了PEG分子量、PEG/(NH4)2SO4质量分数、pH值和温度等因素对双水相成相及人参皂苷萃取的影响。结果表明:PEG分子量为3350、PEG3350的质量分数为12%、(NH4)2SO4质量分数为16%、溶液pH为7.0、温度为60℃时,双水相体系对人参皂苷有较高的萃取率,回收率可到达88.94%。     

不同提取液提取水稻幼苗质外体蛋白效果的比较

提取植物组织质外体蛋白质的主要困难是提取效率低且易被细胞质蛋白污染。为解决上述问题,以12天93-11水稻幼苗为试验材料,使用3种含不同浓度钾和钙离子的缓冲液作为提取液进行提取效果比较。3种提取液的相同成分都是0.1mol/L Tris-HCl pH 7.6,1mmol/L PMSF,区别点在于:Buffer A含0.2mol/L KCl;Buffer B含0.2mol/L CaCl₂;Buffer C含0.1mol/L KCl和0.1mol/L CaCl₂。结果表明,Buffer A的蛋白产率达到了(0.49±0.07)mg/g FW(叶片)和(0.83±0.06)mg/g FW(根部),比Buffer B和Buffer C分别提高了122.7%和53.1%(叶片)以及102.4%和59.6% (根部)。六磷酸葡萄糖脱氢酶活性检测的结果表明在这些蛋白质提取物中细胞质蛋白的污染率很低,可以控制在1%以下。这些实验结果说明通过优化提取液,建立了有效提取水稻幼苗质外体蛋白质的方法,可应用于植物质外体蛋白质组学研究。     

双水相体系萃取分离杜仲叶中桃叶珊瑚甙的研究

建立了由高分子化合物聚乙二醇(PEG4000)与葡聚糖40000(D40)形成的双水相体系萃取分离杜仲叶中桃叶珊瑚甙的新方法.考察了萃取体系相图,研究了PEG4000/D40质量分数、样品溶液加入量、pH值和温度等因素对双水相成相及桃叶珊瑚甙萃取率的影响.结果表明:PEG4000的质量分数为11%,D40质量分数为8%、样品溶液加入量为8 g,温度为60℃,溶液pH为7时,双水相体系对桃叶珊瑚甙有较高的萃取率,重复三次可达到66.32%,而且萃取得到的桃叶珊瑚甙产品的纯度达到48.67%,远远高于粗提物中的8.750%.     

双水相同时提取次烟叶中茄尼醇和烟碱的研究

研究新型双水相体系同时从次等烟丝中提取茄尼醇和烟碱的可行性和工艺条件。新型双水相体系为丙酮-磷酸氢二钾双水相体系,主要研究了丙酮与水不同体积比、磷酸氢二钾的加入量、料液比、浸提温度、浸提时间、双水相体系pH值对茄尼醇和烟碱得率的影响,确定了最佳提取条件:丙酮与水的体积为5∶5,磷酸氢二钾的加入量为3.0 g,料液比为0.025 g/mL(0.5 g/20.0 mL),浸提温度为50℃,浸提时间为4 h,pH值为10。     

Optimization of a Digoxigenin-Based Immunoassay System for Gene Detection in Arabidopsis thaliana

Digoxigenin is derived from a plant steroid hormone digoxin found in the plants Digitalis sp. Digoxigenin has been used successfully in labeling nucleic acids. In this experiment we optimized minimum probe requirement for a nonradioactive digoxigenin-based gene detection system in the model plant Arabidopsis thaliana. We showed that 1 μL of labeled probe was sufficient to hybridize onto 1–10 μg of target plasmid DNA. We also examined the sensitivity of labeled probe and showed that 2 μL of labeled probe was not able to hybridize with 1 μg of target DNA, although 2 μL of labeled probe was able to detect target DNA ranging from 2 to 10 μg. To test the efficacy of our optimization protocol, we used 1 μL of labeled plasmid DNA pU16893 harboring an Arabidopsis housekeeping gene elongation factor-1 and showed that the elongation factor-1 gene could be detected in Arabidopsis genome under various environmental conditions. This paper describes a nonradioactive in situ hybridization technique to detect nucleic acids in plants.     

白地霉脂肪酶的双水相萃取和反胶团提取

对影响双水相萃取和反胶团提取脂肪酶的各种因素进行了探讨,并通过正交实验进一步优化提取条件,PEG浓度15%,(NH4)2SO4浓度22.5%,pH8.0的条件下进行双水相萃取,脂肪酶纯化倍数达到7.5倍;CTAB浓度150mmol/L,相体积比4/2,水相pH8.0,温度40℃的条件下进行反胶团提取,脂肪酶的比活力达到最大,但其比活力稍有下降,约为原来的0.9倍。     

双水相萃取丽江山慈菇中的秋水仙碱

建立了由聚乙二醇(PEG6000)与(NH4)2SO4形成的双水相体系萃取丽江山慈菇中秋水仙碱的新方法。考察了PEG分子量、PEG的浓度、(NH4)2SO4的浓度和pH值对双水相成相及秋水仙碱萃取率的影响,并结合HPLC对萃取相进行检测。结果表明:PEG6000质量分数为8%,(NH4)2SO4质量分数为20%,pH为7.0时,双水相体系对丽江山慈菇粗提液中秋水仙碱萃取率达82.09%,富集倍数为6.84倍。此方法可用于丽江山慈菇中秋水仙碱的初步分离富集,且操作简单,绿色无污染。     

Purification of a Bacteriocin-Like Inhibitory Substance Derived from Pediococcus acidilactici Kp10 by an Aqueous Micellar Two-Phase System

Aqueous micellar two-phase system (AMTPS) is an extractive technique of biomolecule, where it is based on the differential partitioning behavior of biomolecule between a micelle-rich and a micelle-poor phase. In this study, an AMTPS composed of a nonionic surfactant, Triton X-100 (TX-100) was used for purifying a bacteriocin-like inhibitory substance (BLIS) derived from Pediococcus acidilactici Kp10. The influences of the surfactant concentration and the effect of additives on the partitioning behavior and activity yield of the BLIS were investigated. The obtained coexistence curves showed that the mixtures of solutions composed of different surfactant concentrations (5–30% w/w) and 50% w/w crude load were able to separate into two phases at temperatures of above 60 °C. The optimum conditions for BLIS partitioning using the TX-100-based AMTPS were: TX-100 concentration of 22.5% w/w, CFCS load of 50% w/w, incubation time of 30 min at 75 °C, and back-extraction using acetone precipitation. This optimal partitioning resulted in an activity yield of 64.3% and a purification factor of 5.8. Moreover, the addition of several additives, such as sorbitol, KCl, dioctyl sulfosuccinate sodium salt, and Coomassie® Brilliant Blue, demonstrated no improvement in the BLIS separation, except for Amberlite® resin XAD-4, where the activity yield was improved to 70.3% but the purification factor was reduced to 2.3. Results from this study have demonstrated the potential and applicability of TX-100-based AMTPS as a primary recovery method for the BLIS from a complex fermentation broth of P. acidilactici Kp10. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2719, 2019     

氯霉素酶联免疫检测方法的研究

通过活化琥珀氯霉素的羧基使其与蛋白联结合成了免疫抗原 ,并制备了氯霉素的多克隆抗体 ,建立了氯霉素直接竞争酶联免疫检测方法 ,检测时间在 1h之内。抗体的ELISA效价达 3.2× 10 5以上 ,I50 为 12 .9ng ml,检测范围为 0 .2 4ng ml- 6 78.8ng ml。抗体与琥珀氯霉素的交叉反应率为 174 .5 4 % ,与其它抗生素的交叉反应率均小于 0 .0 1%。牛奶样品中平均添加回收率为10 6 .5 6 %。所建立的氯霉素酶联免疫检测方法达到了我国牛奶氯霉素残留限量 0 .2 5ng ml的要求 ,为国产的商品化氯霉素酶免疫检测试剂盒的研制奠定了基础