Essential role of Ubr11, but not Ubr1, as an N‐end rule ubiquitin ligase in Schizosaccharomyces pombe

The N‐end rule pathway degrades proteins bearing a destabilization‐inducing amino acid at the N‐terminus. In this proteolytic system, Ubr ubiquitin ligases recognize and ubiquitylate substrates intended for degradation. Schizosaccharomyces pombe has two similar Ubr proteins, Ubr1 and Ubr11. Both proteins have unique roles in various cellular processes, although the ubr1? strain shows more severe defects. However, their involvement in the N‐end rule pathway is unclear, and even the N‐end rule pathway‐dependent proteolytic activity has not been demonstrated in Sz. pombe. Here, we show that: (a) Sz. pombe has the N‐end rule pathway in which only Ubr11, but not Ubr1, is responsible; and (b) the C‐terminal fragment of the meiotic cohesin Rec8 (denoted as Rec8c) generated by separase‐mediated cleavage is an endogenous substrate of the N‐end rule pathway. Forced overexpression of stable Rec8c was deleterious in mitosis and caused a loss of the mini‐chromosome. In unperturbed mitosis without overexpression, the rate of mini‐chromosome loss was five‐fold higher in the ubr11? strain. Since Rec8 is normally produced in meiosis, we examined whether meiosis and sporulation were affected in the ubr11? strain. In unperturbed meiosis, chromosome segregation occurred almost normally and viable spores were produced in the ubr11? cells, irrespective of the presence of undegraded endogenous Rec8c peptides. Copyright © 2012 John Wiley & Sons, Ltd.     

Fluorescent quantitation method for differentiating the nativity of green fluorescent protein

A gel imaging method was employed to quantitate the GFP that had been subjected to denaturation and degradation treatments. This method is able to differentiate the nativity of GFP by relating the observed changes in the position of fluorescent bands which is unable to be detected using the spectrofluorometric method.     

Genetically modified Lactococcus lactis producing a green fluorescent protein–bovine lactoferrin fusion protein suppresses proinflammatory cytokine expression in lipopolysaccharide-stimulated RAW 264.7 cells

Lactoferrin (LF), an iron-binding glycoprotein distributed widely in the biological fluids of mammals, is believed to play an important role in host defenses against infection. Previous studies in animal models and humans demonstrated that combined administration of LF and probiotic lactic acid bacteria (LAB) can prevent sepsis. In this study, we genetically engineered a probiotic LAB strain, Lactococcus lactis, to produce recombinant bovine LF based on the green fluorescent protein (GFP)-fused expression system. Western blotting confirmed that the genetically modified L. lactis strain (designated NZ-GFP-bLF) produced a protein corresponding to a fusion of GFP and bLF in the presence of nisin, an inducer of target gene expression. The protein synthesized by NZ-GFP-bLF was fluorescent and thus we monitored the time-dependent change in the production level of the recombinant protein using fluorometric analysis. The utility of NZ-GFP-bLF in preventing sepsis was determined by investigating its anti-inflammatory property in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells. Pretreatment of RAW 264.7 cells with NZ-GFP-bLF significantly attenuated the LPS-induced mRNA expression and protein production of 3 proinflammatory cytokines (IL-1α, IL-6, and tumor necrosis factor-α) compared with pretreatment with a vector control strain of L. lactis. Our results suggest that NZ-GFP-bLF holds promise for the development of a new prophylaxis for sepsis.     

报告基因的应用研究进展

目前报告基因作为一种有效技术已在农学、生物医学、生命科学和环境科学等领域起着重要的作用。现以荧光素酶和绿色荧光蛋白为例，综述报告基因的新近应用研究进展。     

报告基因的应用研究进展

目前报告基因作为一种有效技术已在农学、生物医学、生命科学和环境科学等领域起着重要的作用。现以荧光素酶和绿色荧光蛋白为例,综述报告基因的新近应用研究进展。     

天然低共熔溶剂对绿色荧光蛋白的保护作用

天然低共熔溶剂(natural deep eutectic solvent,NADES)是一种新型的绿色环保溶剂,可应用于天然活性物质的提取及生物催化。本研究以绿色荧光蛋白(green flurescent protein,GFP)为模式蛋白,探究了不同NADES对GFP在70℃下的稳定性的影响,发现氯化胆碱-山梨醇(Choline chloride sorbital,CS)对GFP在70℃的热处理下荧光保留率最好;并探究了不同含水量的CS天然低共熔溶剂对GFP稳定性的影响,发现在含水量为40%的CS天然低共熔溶剂中GFP的稳定性最佳;同时发现在含水量为40%的CS天然低共熔溶剂体系下的GFP可以提高对SDS的耐受性,但对H2O2的耐受性的提高不明显;CS天然低共熔溶剂也有利于提高脂肪酶AOL在45℃下的的热稳定性。综上CS天然低共熔溶剂对蛋白质有良好的保护作用,在食品加工及生物催化领域具有潜在的应用前景。     

半纤维素荧光探针的构建与应用

该研究将绿色荧光蛋白（GFP）同木聚糖酶底物结合域（XBD）结合,构建了能够特异性结合半纤维素的荧光探针,经激光扫描共聚焦显微镜比较分析GFP和GFP-XBD荧光融合蛋白对玉米秸秆的结合差异,发现GFP-XBD荧光融合蛋白溶液处理玉米秸秆半纤维素所在区域荧光信号强度明显高于其他样品,表明所构建的半纤维素荧光探针可以与细胞壁中半纤维素特异性结合。荧光测定结果显示,GFP和GFP-XBD的蛋白含量与荧光强度之间有着良好的线性关系（R12 = 0.994 4,R22 = 0.995 1）,成功地表征了玉米秸秆预处理过程中半纤维素的变化规律,最终确定最佳的预处理温度为130 ℃。     

基于转录组分析氯化钙对青圆椒采后机械损伤品质的影响

为探究氯化钙处理对青圆椒采后机械损伤品质的影响,通过高处坠落的方法模拟青圆椒运输过程中受到的机械损伤,利用1 mmol/L氯化钙溶液浸泡处理,测定贮藏期间青圆椒的硬度、色泽及感官品质,并通过高通量测序技术进行转录组分析。结果表明,1 mmol/L氯化钙处理能延缓青圆椒转红、软化及腐烂。同时本研究确定了与青圆椒果实色泽转变、质地变化和风味香气产生的相关基因,鉴定了参与植物激素信号转导途径的关键酶和转录因子,分析了氯化钙处理对机械损伤青圆椒果实贮藏品质的影响。氯化钙可能通过抑制编码双功能15-顺式八氢番茄红素合酶（bifunctional 15-cis-phytoene synthase,PSY）和辣椒红色素/辣椒红色素合酶（capsanthin/capsorubin synthase,CCS）等基因的上调来抑制类胡萝卜素的合成,通过抑制纤维素合酶A（cellulose synthase A,Ces）、内切-1,3-β-葡萄糖苷酶（endo-1,3-β-glucosidase 4,β-Glu）和内切葡聚糖酶25（endoglucanase 25,EG25）等基因的下调来缓解其软化,并通过影响青圆椒中某些转录因子、植物激素和香气风味相关基因的表达,进而影改善有机械损伤的青圆椒果实的表观品质。结论：氯化钙处理可以有效延缓机械损伤青圆椒果实品质的劣变,延长青圆椒果实的贮藏期。     

cA-GFP融合蛋白在大肠杆菌中的高效功能性表达

为了实现乳酸乳球菌锚定蛋白cA在大肠杆菌中可溶表达以及直观检测其生物学活性,以乳酸乳球菌MG1363为模板,扩增N-乙酰葡萄糖胺糖苷酶AcmA基因,将其C末端序列与GFP基因融合,连接至大肠杆菌表达载体pET28a,构建重组质粒pET28a-cA-GFP,转化至大肠杆菌表达菌株BL21(DE3),通过低温诱导表达重组蛋白cA-GFP。工程菌超声破碎后的上清液与经热酸处理的乳酸乳球菌常温孵育,经SDS-PAGE和荧光显微镜检测。结果表明,工程菌可以表达分子量约53ku的目的融合蛋白质,与预期大小相符。cA-GFP通过锚定蛋白cA的引导回向锚定,成功将GFP展示在乳酸乳球菌表面,目的蛋白cA-GFP在乳酸乳球菌表面展示量为121mg/g干重菌体。GFP锚定至乳酸乳球菌后于4℃保存,连续6d测定其荧光强度,荧光强度仍可达82.2%,证明其稳定性较好。成功获得了具有生物学功能的cA-GFP可溶性蛋白,为进一步展示功能性外源蛋白奠定了坚实的基础。     

绿色荧光蛋白的原核表达、纯化与荧光分析

将绿色荧光蛋白（GFP）基因插入到pET30（a）＋构建pET30-GFP表达载体，将重组载体导入大肠杆菌BL-21中，经IPTG诱导产生His—GFP融合蛋白，融合蛋白主要存在于可溶性上清中。用镍柱亲合纯化His—GFP，分子量大约为32．4kD，与预期值相符。荧光分析表明，His—GFP与野生型GFP荧光激发及发射波长基本相同，荧光性质稳定，比常用的荧光素（FITC）具有更强的抗荧光淬灭能力。通过此方法制备的绿色荧光蛋白可用于食用色素的开发等。     

杏鲍菇空气煎炸过程品质变化与羧甲基赖氨酸、羧乙基赖氨酸生成研究

本文以杏鲍菇脆片为研究对象,分别采用荧光光谱法和超高效液相色谱-三重四极杆串联质谱法测定脆片中荧光性晚期糖基化终末产物（Advanced glycation end products,AGEs）以及2种极具代表性的典型晚期糖基化终末产物羧甲基赖氨酸（N^ε-carboxymethyl-lysine,CML）和羧乙基赖氨酸（N^ε-carboxyethyl-lysine,CEL）的含量,并研究不同煎炸温度、时间对杏鲍菇品质的影响。结果表明：杏鲍菇脆片在煎炸过程中发生了褐变,色泽开始由白色变成金黄色,时间越长、温度越高,颜色越深。脆片的水分含量显著（P<0.05）减少,硬度变化趋势为先减小后增大,且在煎炸过程中,煎炸时间和温度对杏鲍菇中CML和CEL的形成均具有显著（P<0.05）的影响,其含量随着煎炸温度和煎炸时间的增加而不断增加。在180 ℃、20 min下,CML和CEL的含量达到了最高,分别是13.75和70.63 μg/g。但荧光AGEs的生成量在180 ℃、10 min要比160 ℃、10 min高4.5 AU。在160 ℃、10 min下,CML和CEL的含量分别是4.54和8.34 μg/g,相比于160 ℃、20 min下,分别减少了46.65%和62.48%,且在此条件下感官评定较好,与180 ℃、10 min差异不显著,同时生成的AGE较少。因此结合各个指标的测定结果和感官评价,最终确定杏鲍菇脆片最佳煎炸条件为160 ℃、10 min。