HPT-mCherry融合标签在水稻转基因筛选鉴定中的应用

目的：利用HPT-mCherry融合标签加速转基因筛选过程。方法：构建HPT-mCherry融合表达载体,采用农杆菌介导的转基因技术获得转基因植株,并利用荧光显微镜观察转基因愈伤和植株。结果：HPT-mCherry融合标签可用于转基因愈伤的筛选和转基因植株的鉴定;HPT与mCherry融合后互不影响,一方面可利用潮霉素抗性筛选愈伤,另一方面能通过观察mCherry红色荧光蛋白进行鉴定。结论：利用HPT-mCherry融合标签可以快速有效地鉴定并得到转基因阳性植株。     

TAT介导多肽药物过表达纯化和穿膜活性检测

目的:获得NR2B羧基端多肽(NR2Pep),探讨TAT-NR2Pep在细胞中的跨膜特性。方法:利用酶切连接方法构建p Waldo-TAT-pep质粒,转化大肠杆菌BL21(DE3)感受态细胞,检测不同浓度诱导剂和不同温度对蛋白质表达的影响;利用镍柱亲和层析及分子筛方法纯化TAT-NR2Pep-GFP融合蛋白,利用GFP发荧光的特性采用In-gel检测蛋白质的表达情况;以融合蛋白C端His标签特异性抗体,通过Western印迹确定融合蛋白的表达;荧光显微镜下观察TAT-NR2Pep-GFP融合蛋白在CHO和C2C12等细胞中的跨膜活性。结果:构建了TAT-NR2Pep-GFP表达载体,重组菌经0.1 mmol/L IPTG于22℃诱导20 h能够表达较多高质量的目标蛋白;In-gel荧光检测证实融合蛋白TATNR2Pep-GFP拥有正确构象,GFP能够发出绿色荧光;Western印迹分析表明融合蛋白相对分子质量符合预期;细胞穿膜实验证实TAT能够介导多肽穿过细胞膜。结论:原核表达并纯化得到TAT-NR2Pep-GFP融合蛋白,该蛋白散发绿色荧光,能够穿过细胞膜。     

Mpl与绿色荧光蛋白融合基因的真核载体构建及表达

目的:探索Mpl与绿色荧光蛋白GFP基因共同转粢哺乳动物细胞NIH3T3的方法.方法:采用PCR方法将GFP基因与Mpl基因构建融合荧光蛋白的真核表达载体,用脂质体介导转染NIH3T3细胞和筛选稳定细胞系,使用荧光显微镜方法和Westernblotting检测转染效果.结果:利用PCR方法有效扩增了Mpl基因,构建了融合荧光蛋白的真核表达载体,序列分析表明所构建的含Mpl基因的质粒与设计相同,使用荧光显微镜方法和Western blotting检测Mpl融合绿色荧光蛋白表达载体成功转染NIH3T3细胞.结论:成功构建了Mpl荧光表达载体,融合基因可以在NIH3T3细胞中稳定表达,为进一步研究Mpl的生物学活性及其与hNUDC蛋白相互作用提供了重要的理论依据.     

GFP-SA融合蛋白的表达纯化及其锚定修饰肿瘤细胞的研究

目的　GFP（绿色荧光蛋白）-SA（链亲和素）双功能融合蛋白的制备及其鉴定研究,以展示我们建立的技术平台,即用含链亲和素的双功能融合蛋白对生物素化的细胞表面进行高效的锚定修饰。方法　构建原核表达载体pET24d/GFP-SA转化大肠杆菌BL21(DE3)。用IPTG诱导重组蛋白的表达,用镍金属螯合（Ni-NTA）层析柱进行纯化。用制备的GFP-SA双功能融合蛋白,对B16肿瘤细胞已生物素化的细胞表面进行修饰,经荧光显微镜和流式细胞仪进行修饰效率分析。此外,用MTT法检测细胞表面修饰对肿瘤细胞活力及其生长情况的影响。结果　GFP-SA重组融合蛋白在大肠杆菌实现了高效表达（约占细菌总蛋白的20%）,通过纯化和复性制备的GFP-SA双功能融合蛋白具有双重活性,即：链亲和素介导的、对生物素高效特异的结合活性,和GFP发射绿色荧光的活性,并能高效修饰表面已生物素化的肿瘤细胞。此外,GFP-SA双功能融合蛋白的细胞表面修饰对细胞的活力及其生长无显著影响。结论　GFP-SA融合蛋白能高效修饰表面已生物素化的肿瘤细胞,可用作肿瘤疫苗研究的示踪蛋白及实验对照体系。     

缓激肽生物合成及其对心肌细胞缺氧复氧保护作用

[目的]获得有活性的缓激肽药物,探讨缓激肽在心肌细胞缺氧复氧过程中的保护作用。[方法]利用PCR方法构建p Waldo-BK质粒,转化大肠杆菌BL21(DE3)表达菌株,优化诱导剂IPTG浓度和表达温度;利用镍柱亲和层析和分子筛纯化缓激肽融合蛋白质,利用GFP荧光In-gel检测蛋白质的表达情况;构建心肌细胞缺氧复氧模型,检测缓激肽对细胞的保护作用。[结果]成功构建p Waldo-BK表达载体,0. 2 mmol/L IPTG于25℃诱导培养20 h,能够获得高表达的目标蛋白质; In-gel荧光检测证实,融合蛋白GFP能够发出绿色荧光;心肌细胞缺氧复氧模型证实缓激肽能够有效保护心肌细胞。[结论]0. 2 mmol/L IPTG、25℃诱导表达20 h,得到散发绿色荧光的融合蛋白,每1L BL21(DE3)细胞培养基能够获得缓激肽约0. 55 mg,且最终获得的缓激肽能够有效保护心肌细胞对抗缺氧复氧刺激。     

绿色荧光蛋白表达载体pXS75－GFP的构建及在嗜热四膜虫中的应用

为获得能够用于构建嗜热四膜虫蛋白定位的载体,该研究将GFP基因与镉（Cd2＋）诱导的四膜虫金属硫蛋白基因（MTTl）启动子序列和终止子序列融合,获得表达载体pXS75-GFP。通过同源重组和抗性筛选,pXS75-GFP载体携带的目的基因整合入四膜虫MTTl位点,在cd2＋诱导下实现GFP融合蛋白的可控表达。将α－tubulin基因ATUl克隆JN－pXS75－GFP中,重组质粒pXS75－GFP－ATUl通过基因枪转化入四膜虫细胞,在巴龙霉素筛选下获得稳定的α-tubulin－GFP过表达细胞株。激光共聚焦显微镜观察α－tubulin．GFP的定位,结果显示,α－tubulin—GFP融合蛋白在四膜虫细胞中表达并分布于皮层上,表明pXS75．GFP载体可用于嗜热四膜虫功能蛋白的定位分析。     

高中综合实验设计——以"表达和纯化红色荧光蛋白标记的类弹性蛋白"为例

以类弹性蛋白(elastin-like polypeptide,ELP)作为非色谱纯化标签,分离纯化红色荧光蛋白 mCherry.ELP 与△ I-CM(intein cleavage mutant)的 N 端连接,mCherry 片段与△I-CM的C端连接,在ELP的N端插入GFP片段,用于检测蛋白纯度.采用低温诱导...     

金黄色葡萄球菌双组分系统反应调节蛋白AgrA的原核表达、纯化及活性鉴定

AgrA作为金黄色葡萄球菌双组分信号转导系统(two-component signal transduction system,TCST)的反应调节因子,能调控细菌毒力因子的表达,在金黄色葡萄球菌致病过程中起着重要的作用。采用无限制克隆法构建AgrA表达载体,在AgrA蛋白的C端融合绿色荧光蛋白(GFP)标签,通过实时监测GFP的荧光强度来快速检测重组蛋白的表达水平。首先利用单因素实验,筛选出宿主菌株BL21-(DE3)-PlysS;其次,结合Box-Behnken试验设计,筛选出最优蛋白质表达条件:诱导时间为22h、转速为222r/min、诱导剂浓度为0.5mmol/L,AgrA产量达到5.56mg/L。最后,基于AgrA蛋白LytTR区域的非放射性凝胶阻滞实验(non-radioactive electrophoretic mobility shift assay,EMSA)验证了AgrA的生物活性。提出了反应调节蛋白AgrA在大肠杆菌高效可溶性表达的策略,为双组分信号转导系统的体外研究奠定基础,也为其他反应调节蛋白的可溶性表达与分离纯化提供了一个可行借鉴。     

动态检测活细胞内泛素-蛋白酶体系统活性方法的建立

目的建立一种动态检测活细胞内泛素-蛋白酶体系统活性的方法。方法将表达绿色荧光蛋白（GFP）或红色荧光蛋白（DsRed2）的质粒分别改建为表达带有内泛素-蛋白酶体系统降解信号CL1的GFP或DsRed2的pGFP^u或pDsRed2质粒,然后转染HEK293细胞,通过G418筛选得到稳定表达GFP^u或DsRed2^u的细胞系。在蛋白酶体抑制N—Acetyl—Leu-Leu—Norleu—al（ALLN）处理GFP^u或DsRed2^u细胞后,应用免疫印记技术检测细胞内GFP或DsRed,含量的变化,应用荧光显微镜和激光扫描共聚焦显微镜技术观察GFP或DsRed,荧光强度的变化。结果ALLN处理能使GFP“和DsRed2^u细胞内GFP和DsRed。含量明显增加,荧光强度显著增强,并呈现明显的剂量／时间-效应关系。结论本文成功地建立了检测内泛素-蛋白酶体系统活性的方法,该方法能有效地对活细胞的内泛素-蛋白酶体系统活性进行实时动态检测。     

慢病毒载体系统介导hUC—MSC绿色荧光蛋白和荧光素酶共表达技术体系的建立

目的建立慢病毒载体系统介导的人脐带间充质干细胞（hUC-MSC）绿色荧光蛋白（GFP）和荧光素酶共表达技术体系。方法 GFP和荧光素酶共表达慢病毒载体与相应包装质粒psPAX2和pMD2.G经聚乙烯亚胺介导共转染HEK293T细胞以包装病毒;病毒感染P4代hUC-MSC 12 h后,再行嘌呤霉素筛选24 h,普通光学显微镜观察细胞形态,荧光显微镜下观察GFP的表达情况,IVIS Kinetic成像系统拍照以观察和记录慢病毒感染后hUC-MSC荧光素酶的表达情况;MTS法行细胞生长曲线作图,同时,普通和实时定量RTPCR法检测细胞周期调控相关蛋白Cyclin D1、Cyclin E1和p21WAF1/CIP1的表达。采用方差分析和t检验进行统计学分析。结果慢病毒感染并不会造成体外培养hUC-MSC形态的明显改变,而荧光显微镜和IVIS Kinetic成像系统的观察结果则分别证实,GFP和荧光素酶经慢病毒载体系统的介导可在hUC-MSC中成功地共表达。此外,细胞生长曲线作图结果表明,对照和GFP及荧光素酶共表达慢病毒感染后hUC-MSC的生长增殖速率相仿（P〉0.01）;实时定量RT-PCR法检测结果则显示,与对照慢病毒感染相比,GFP和荧光素酶共表达慢病毒感染后其细胞周期调控相关蛋白Cyclin D1、Cyclin E1和p21WAF1/CIP1mRNA表达水平分别是对照组的1.11倍（P=0.130）、0.54倍（P=0.000）和0.78倍（P=0.005）,表明外源GFP和荧光素酶共表达对体外培养的hUC-MSC生长增殖等表型无显著影响。结论慢病毒载体系统可有效介导外源基因在hUC-MSC中的表达;同时,GFP和荧光素酶在hUC-MSC中的共表达也将极大地方便其体内转归的示踪。     

Discovery of a new class of PROTAC BRD4 degraders based on a dihydroquinazolinone derivative and lenalidomide/pomalidomide

BRD4 has emerged as an attractive target for anticancer therapy. However, BRD4 inhibitors treatment leads to BRD4 protein accumulation, together with the reversible nature of inhibitors binding to BRD4, which may limit the efficacy of BRD4 inhibitors. To address these problems, a protein degradation strategy based on the proteolysis targeting chimera (PROTAC) technology has been developed to target BRD4 recently. Herein, we present our design, synthesis and biological evaluation of a new class of PROTAC BRD4 degraders, which were based on a potent dihydroquinazolinone-based BRD4 inhibitor compound 6 and lenalidomide/pomalidomide as ligand for E3 ligase cereblon. Gratifyingly, several compounds showed excellent inhibitory activity against BRD4, and high anti-proliferative potency against human monocyte lymphoma cell line THP-1. Especially, compound 21 (BRD4 BD1, IC₅₀ = 41.8 nM) achieved a submicromolar IC₅₀ value of 0.81 μM in inhibiting the growth of THP-1 cell line, and was 4 times more potent than compound 6. Moreover, the mechanism study established that 21 could effectively induce the degradation of BRD4 protein and suppression of c-Myc. All of these results suggested that 21 was an efficacious BRD4 degrader for further investigation.     

Impact of linker length on the activity of PROTACs

Conventional genetic approaches have provided a powerful tool in the study of proteins. However, these techniques often preclude selective manipulation of temporal and spatial protein functions, which is crucial for the investigation of dynamic cellular processes. To overcome these limitations, a small molecule-based novel technology termed "PROteolysis TArgeting ChimeraS (PROTACs)" has been developed, targeting proteins for degradation at the post-translational level. Despite the promising potential of PROTACs to serve as molecular probes of complex signaling pathways, their design has not been generalized for broad application. Here, we present the first generalized approach for PROTAC design by fine-tuning the distance between the two participating partner proteins, the E3 ubiquitin ligase and the target protein. As such, we took a chemical approach to create estrogen receptor (ER)-α targeting PROTACs with varying linker lengths and the loss of the ER in cultured cells was monitored via western blot and fluorometric analyses. We found a significant effect of chain length on PROTAC efficacy, and, in this case, the optimum distance between the E3 recognition motif and the ligand was a 16 atom chain length. The information gathered from this experiment may offer a generalizable PROTAC design strategy to further the expansion of the PROTAC toolbox, opening new possibilities for the broad application of the PROTAC strategy in the study of multiple signaling pathways.     

The development, characterization, and demonstration of a novel strategy for purification of recombinant proteins expressed in plants

Plants have attracted increasing attention as an expression platform for the production of pharmaceutical proteins due to its unlimited scalability and low cost potential. However, compared to other expression systems, plants accumulate relatively low levels of foreign proteins, thus necessitating the development of efficient systems for purification of foreign proteins from plant tissues. We have developed a novel strategy for purification of recombinant proteins expressed in plants, based on genetic fusion to soybean agglutinin (SBA), a homotetrameric lectin that binds to N-acetyl-D-galactosamine. Previously it was shown that high purity SBA could be recovered from soybean with an efficiency of greater than 90% following one-step purification using N-acetyl-D-galactosamine-agar columns. We constructed an SBA fusion protein containing the reporter green fluorescent protein (GFP) and transiently expressed it in N. benthamiana plants. We achieved over 2.5% of TSP accumulation in leaves of N. benthamiana. Confocal microscopic analysis demonstrated in vivo activity of the fused GFP partner. Importantly, high purity rSBA-GFP was recovered from crude leaf extract with ~90% yield via one-step purification on N-acetyl-D-galactosamine-agar columns, and the purified fusion protein was able to induce the agglutination of rabbit red blood cells. Combined with this, tetrameric assembly of the fusion protein was demonstrated via western blotting. In addition, rSBA-GFP retained its GFP signal on agglutinated red blood cells, demonstrating the feasibility of using rSBA-GFP for discrimination of cells that bear the ligand glycan on their surface. This work validates SBA as an effective affinity tag for simple and rapid purification of genetically fused proteins.     

Biosynthesis and metabolic engineering of 1-hydroxyphenazine in Pseudomonas chlororaphis H18

The split GFP assay is a well-known technology for activity-independent screening of target proteins. A superfolder GFP is split into two non-fluorescent parts, GFP11 which is fused to the target protein and GFP1-10. In the presence of both, GFP1-10 and the GFP11-tag are self-assembled and a functional chromophore is formed. However, it relies on the availability and quality of GFP1-10 detector protein to develop fluorescence by assembly with the GFP11-tag connected to the target protein. GFP1-10 detector protein is often produced in small scale shake flask cultivation and purified from inclusion bodies. The production of GFP1-10 in inclusion bodies and purification was comprehensively studied based on Escherichia coli as host. Cultivation in complex and defined medium as well as different feed strategies were tested in laboratory-scale bioreactor cultivation and a standardized process was developed providing high quantity of GFP1-10 detector protein with suitable quality. Split GFP assay was standardized to obtain robust and reliable assay results from cutinase secretion strains of Corynebacterium glutamicum with Bacillus subtilis Sec signal peptides NprE and Pel. Influencing factors from environmental conditions, such as pH and temperature were thoroughly investigated. GFP1-10 detector protein production could be successfully scaled from shake flask to laboratory scale bioreactor. A single run yielded sufficient material for up to 385 96-well plate screening runs. The application study with cutinase secretory strains showed very high correlation between measured cutinase activity to split GFP fluorescence signal proofing applicability for larger screening studies.     

Chemical genetics approaches for selective intervention in epigenetics

In conventional chemical genetics, cell-active small-molecules directly block protein activity, altering phenotype. However these molecules may not be sufficiently selective or effective at modulating complex epigenetic pathways. By mutating the target protein, and creating a mutant-selective inhibitor, the bump-and-hole approach can provide single-target selectivity. PROTAC molecules direct their target to proteosomal degradation by recruiting an E3 ubiquitin ligase, resulting in more efficacious target downregulation.

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靶向蛋白质泛素修饰及降解在前列腺癌治疗中的机遇与挑战

前列腺癌是中国发病率增长最快的男性肿瘤,抗雄激素治疗耐药是导致前列腺癌患者预后差的主要原因。因此,解决耐药性难题是前列腺癌转化研究的关键问题。哺乳动物细胞利用泛素-蛋白酶体系统实现蛋白质的靶向降解。因此,前列腺癌中关键的癌基因如雄激素受体（AR）的上游泛素化调控因子（如去泛素化酶）是潜在的治疗靶点。然而,这些酶具有较广的底物谱系,存在脱靶的可能性。近来,基于泛素-蛋白酶体系统开发的蛋白质降解靶向嵌合体（proteolysis-targeting chimeras,PROTAC）技术是最具前景和革命性的新型抗癌药物研发技术,能够利用特定E3泛素连接酶对靶蛋白进行降解而不影响其他底物。与传统小分子抑制剂相比,PROTAC分子在克服耐药性以及针对不可成药的靶点方面拥有巨大优势。目前,针对AR的PROTAC降解剂已在II期临床取得了成功,靶向蛋白质泛素化及降解途径的新技术将有望为前列腺癌的临床治疗带来新的突破。     

Protein Evolution via Amino Acid and Codon Elimination

Background

Global residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained via screening of reduced-size ensembles.

Methodology/Principal Findings

The strategy involves combining a sequential mutagenesis scheme to reduce library size with structurally stabilizing mutations, chaperone complementation, and reduced temperature of gene expression. In six steps, we eliminated a common buried residue, Phe, from the green fluorescent protein (GFP), while retaining activity. A GFP variant containing 11 Phe residues was used as starting scaffold to generate 10 separate variants in which each Phe was replaced individually (in one construct two adjacent Phe residues were changed simultaneously), while retaining varying levels of activity. Combination of these substitutions to generate a Phe-free variant of GFP abolished fluorescence. Combinatorial re-introduction of five Phe residues, based on the activities of the respective single amino acid replacements, was sufficient to restore GFP activity. Successive rounds of mutagenesis generated active GFP variants containing, three, two, and zero Phe residues. These GFPs all displayed progenitor-like fluorescence spectra, temperature-sensitive folding, a reduced structural stability and, for the least stable variants, a reduced steady state abundance.

Conclusions/Significance

The results provide strategies for the design of novel GFP reporters. The described approach offers a means to enable engineering of active proteins that lack certain amino acids, a key step towards expanding the functional repertoire of uniquely labeled proteins in synthetic biology.     

Membrane Topology and Cellular Dynamics of Foot-and-Mouth Disease Virus 3A Protein

Foot-and-mouth disease virus non-structural protein 3A plays important roles in virus replication, virulence and host-range; nevertheless little is known on the interactions that this protein can establish with different cell components. In this work, we have performed in vivo dynamic studies from cells transiently expressing the green fluorescent protein (GFP) fused to the complete 3A (GFP3A) and versions including different 3A mutations. The results revealed the presence of a mobile fraction of GFP3A, which was found increased in most of the mutants analyzed, and the location of 3A in a continuous compartment in the cytoplasm. A dual behavior was also observed for GFP3A upon cell fractionation, being the protein equally recovered from the cytosolic and membrane fractions, a ratio that was also observed when the insoluble fraction was further fractioned, even in the presence of detergent. Similar results were observed in the fractionation of GFP3ABBB, a 3A protein precursor required for initiating RNA replication. A nonintegral membrane protein topology of FMDV 3A was supported by the lack of glycosylation of versions of 3A in which each of the protein termini was fused to a glycosylation acceptor tag, as well as by their accessibility to degradation by proteases. According to this model 3A would interact with membranes through its central hydrophobic region exposing its N- and C- termini to the cytosol, where interactions between viral and cellular proteins required for virus replication are expected to occur.     

Hi-JAK-ing the ubiquitin system: The design and physicochemical optimisation of JAK PROTACs

PROTACs have recently emerged as a novel paradigm in drug discovery. They can hijack existing biological machinery to selectively degrade proteins of interest, in a catalytic fashion. Here we describe the design, optimisation and biological activity of a set of novel PROTACs targeting the Janus kinase family (JAK1, JAK2, JAK3 and TYK2) of proximal membrane-bound proteins. The JAK family proteins display membrane localisation by virtue of their association with cytoplasmic tails of cytokine receptors, and there are no reports of a successful PROTAC strategy being deployed against this class of proteins. JAK PROTACs from two distinct JAK chemotypes were designed, optimising the physicochemical properties for each template to enhance cell permeation. These PROTACs are capable of inducing JAK1 and JAK2 degradation, demonstrating an extension of the PROTAC methodology to an unprecedented class of protein targets. A number of known ligase binders were explored, and it was found that PROTACs bearing an inhibitor of apoptosis protein (IAP) ligand induced significantly more JAK degradation over Von Hippel–Lindau (VHL) and Cereblon (CRBN) PROTACs. In addition, the mechanism of action of the JAK PROTACs was elucidated, and it was confirmed that JAK degradation was both IAP- and proteasome-dependent.     

Assessment of membrane protein expression and stability using a split green fluorescent protein reporter

Membrane proteins are challenging targets for structural biologists. Finding optimal candidates for such studies requires extensive and laborious screening of protein expression and/or stability in detergent. The use of green fluorescent protein (GFP) as a reporter has enormously facilitated these studies; however, its 238 residues can potentially alter the intrinsic properties of the target (e.g., expression or stability). With the aim of minimizing undesired effects of full-length GFP, here we describe the utility of a split GFP reporter during precrystallization studies of membrane proteins. GFP fluorescence appeared by complementation of the first 15 residues of GFP (GFP(11)) (fused to the C terminus of a membrane protein target) with the remaining nonfluorescent GFP (GFP(1-10)). The signal obtained after sequential expression of SteT (l-serine/l-threonine exchanger of Bacillus subtilis) fused to GFP(11) followed by GFP(1-10) specifically measured the protein fraction inserted into the Escherichia coli cytoplasmic membrane, thereby discarding protein aggregates confined as inclusion bodies. Furthermore, in vitro complementation of purified SteT-GFP(11) with purified GFP(1-10) was exploited to rapidly assess the stability of wild-type and G294V mutant versions of SteT-GFP(11) following detergent solubilization and purification. This method can be applied in a medium- to high-throughput manner with multiple samples.