Analysis of Protein Interactions:Probing the Function of Proteins with Yeast Two-Hybrid System

The yeast two\|hybrid system is a molecular genetic approach for protein interaction and it is widely used to screen for proteins that interact with a protein of interest in recent years.This process includes,construction and testing of the bait plasmid,screening a plasmid library for interacting fusion protein,elimination of false positives and delection analysis of true positives.This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with a protein of interest.More and more studies have demonstrated that the two\|hybrid system is a powerful and sensitive technique for the identification of genes that code for proteins that interact in a biologically significant fashion with a protein of interest in higher plants.This method has been used to identify new interaction protein in many laboratories.The recently reported yeast tri\|brid system,should allow the investigation of more complex protein\|protein interactions.The aim of this review is to outline the recent progress made in protein interactions by using yeast two\|hybrid system.

Screening for Novel Binding Proteins Interacting with Human Papillomavirus Type 18 E6 Oncogene in the Hela cDNA Library by Yeast Two-Hybrid System

To screen for novel binding proteins interacting with high-risk HPV 18 E6 oncogene, the strain AH 109 was transformed with pGBKT7-HPV 18 E6 plasmid, and subsequent transference was utilized to screen for interacting proteins with HPV 18 E6 in human Hela cDNA library. HPV 18 E6 mRNA was expressed in yeast and there was no self-activation and toxicity in AH109. Seven proteins that interacted with HPV18 E6, including transmembrane protein 87B, phosphonoformate immuno-associated protein 5, vimentin, KM-HN-1 protein, dedicator of cytokinesis 7, vaccinia related kinase 2 and a hypothetical protein, were identified. It was suggested that yeast two-hybrid system is an efficient for screening interacting proteins. The high-risk HPV 18 E6 oncogene may interact with the proteins, which may be associated with signal transduction and transcriptional control, epithelial cell invasion and migration, as well as humoral and cellular immune etc. This investigation provides functional clues for further exploration of potential oncogenesis targets for cancer biotherapy.

Shared and discrete interacting partners of ELL1 and ELL2 by yeast two-hybrid assay

ELL2 (eleven-nineteen lysine-rich leukemia transcription elongation factor), a component of a larger complex with pTEFb (cyclin T and CDK9) and AF4, is up-regulated in plasma cells where it influences mRNA processing by increasing exon skipping and enhancing proximal poly (A) site use. ELL2 is needed to produce the secretory-specific Ig heavy chain mRNA while ELL1 mRNA does not change in abundance with B cell stages. To investigate the potential interactions of other proteins with the ELL1 and ELL2 proteins, we preformed yeast two-hybrid studies. HSP40 and Testin were found to bind to ELL2 in its amino-terminal half. PCNA binds to ELL2 in a region encompassing amino acids 186 - 344. The potent transcription factors HIF1 α and ZNF622 interact with both ELL1 and 2 in the central, proline rich region. Meanwhile, BBS2 and ING3 interact with ELL1 but not ELL2 in this central proline-rich region. Many of the ELL-interacting-proteins uncovered in the two-hybrid screen are tumour suppressors that may work through the ELL: pTEFb complex to suppress or activate sets of genes in plasma cells.

Screening proteins that interact with mutant superoxide dismutase 1 from familial amyotrophic lateral sclerosis using a yeast two-hybrid system

The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 （SOD1） using a yeast two-hybrid system. Using BLAST software, 15 real proteins which interacted with mutant SOD1 were obtained, including 8 known proteins （protein tyrosine-phosphatase non-receptor type 2, TBCl D4, protein kinase family, splicing factor, arginine/serine-rich 2, SRC protein tyrosine kinase Fyn, β-sarcoglycan; glycine receptor a2, microtubule associated protein/microtubule affinity-regulating kinase 1, ferritin H chain）, and 7 unknown proteins. Results demonstrated interaction of mutant SOD1 with microtubule associated protein/microtubule affinity-regulating kinase 1 and β-sarcoglycan.

Construction and Identification of a Yeast Two-Hybrid Bait Vector and Its Effect on the Growth of Yeast Cells and the Self-Activating Function of Reporter Genes for Screening of HPV18 E6-Interacting Protein

By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of reporter genes were investigated.Total mRNA extracted from Hela cells was reversely transcribed into cDNA.Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector.The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.Th...

Construction of a Three-frame Yeast Two-hybrid cDNA Library of Fusarium oxysporum

A specialized test of two-hybrid library type three-frame cDNA yeast for Muskmelon Fusarium oxysporum using the switching mechanism at the 5'end of RNA template(SMART)technology was constructed to screen for interaction protein genes for wilt disease and to further research the molecular mechanisms of Fusarium oxysporum pathogenesis to explain the interactions between plant and pathogen.A 500-bp cDNA was purified and extracted using SMART and LD-PCR technology to synthesize ds cDNA and was then homogenized and purified to remove the fragments.After processing,the ds cDNA was connected to three types of reading frame pGADT7-SfiI carriers,and the three connection products in E.coli Electrocell were used to build the primary cDNA library.The titer of three ORF cDNA primary library storage capacities was 2.6×10^6,1.8×10^6 and 3×10^6 cfu;the PCR identification of the ORF 1 and 2 gene recombination rate was 94%,the ORF 3 gene recombination rate was 100%,and the insert length distribution was 0.5-4.0 kb as a single band.To reach the quality requirements for library construction,three kinds of reading frame cDNA primary libraries were mixed and amplified,and the plasmid was transformed into the Y187 yeast strain.The titer of the Y187 yeast library was determined to be 3.5×107 cfu?mL-1,and the base of the yeast library was approximately 1 600 000 cfu.The results showed that the construction of muskmelon Fusarium-specific two-hybrid library type three-frame cDNA yeast had a higher reservoir capacity and recombination rate and met the yeast two-hybrid screening requirements.

Screening of Host Proteins Interacting with PorcineEpidemic Diarrhea Virus (PEDV) N Protein by YeastTwo-hybrid System

[Objective] The paper was to obtain host proteins interacting with porcine epidemic diarrhea virus （PEDV） N protein. [Method] The re-combinant vector pGBKT7-N of PEDV N gene was constructed and used as the bait plasmid to screen the proteins interacting with N protein ofPEDV from the cDNA library of porcine alveolar macrophage （PAM） by yeast two-hybrid method. [Result] There was no toxicity and self activationof bait protein in yeast hybridization system, and six proteins （FTH1, LGALS3, CORO1C, SNRPG, KRTAP5-3, ZNF598） interacting with N proteinwere indentified. It was confirmed that LGALS3 and SNRPG had specific interaction with N protein by return experiment and co-immunoprecipitation（CoIP） test. [Conclusion] The study lays a foundation for further studying the function of PEDV N protein and the pathogenic mechanism of PEDV.

Screening of hepatocyte proteins binding to NS5ABP37 protein by yeast-two hybrid system

Objective To investigate the biological function of NS5ABP37 and to look for proteins interacting with NS5ABP37 protein in hepatocytes.Methods We constructed bait plasmid expressing NS5ABP37 protein of hepatitis C virus(HCV)by cloning the gene of NS5ABP37 protein into pGBKT7,then the recombinant plasmid DNA was transformed into yeast AH109(α type).The transformed yeast AH109 was mated with yeast Y187(α type)containing liver cDNA library plasmid in 2×YPDA medium.Diploid yeast was plated on synthetic dropout nutrient medium(SD/-Trp-Leu-His-Ade)containing X-α-gal for selection and screening.After extracting and sequencing of plasmids from positive(blue)colonies,we made a sequence analysis by bioinformatics.Results We screened twenty-five proteins binding to NS5ABP37,including Homo sapiens cyclin I(CCNI)gene,Homo sapiens matrix metallopeptidase 25(MMP25)and Homo sapiens talin 1.Conclusion The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with NS5ABP37 of HCV.And the biological function of NS5ABP37 may be associated with glycometabolism,lipid metabolism and apoptosis.

Yeast One-hybrid System Used to Identify the Binding Proteins for Rat Glutathione S-transferase P Enhancer I

Objective To detect the trans-factors specifically binding to the strong enhancerelement (GPEI) in the upstream of rat glutathione S-transferase P (GST-P) gene. MethodsYeast one-hybrid system was used to screen rat lung MATCHMAKER cDNA library toidentify potential trans-factors that can interact with core sequence of GPEI(cGPEI).Electrophoresis mobility shift assay (EMSA) was used to analyze the binding of trans-factors to cGPEI. Results cDNA fragments coding for the C-terminal part of thetranscription factor c-Jun and rat adenine nucleotide translocator (ANT) were isolated. Thebinding of c-Jun and ANT to GPEI core sequence were confirmed. Conclusions Rat c-juntranscriptional factor and ANT may interact with cGPEI. They could play an important rolein the induced expression of GST-P gene.

Improved methods for cloning and detection in the yeast two hybrid assay

The yeast two-hybrid (Y2H) mating assay is a powerful method for detecting protein-protein interactions. Firstly, the gene of interest is cloned into specific Y2H vectors. Although multiple innovations in cloning methods were made in the past two decades, the conventional cloning method of restriction-enzyme (RE) digestion followed by ligation is still widely used. Unfortunately, many researchers, especially new-comers, often encounter difficulties in cloning a gene into a desired vector. Secondly, interaction between two proteins is commonly detected by growth of the diploids in specific media. This step takes about two weeks. Here, we describe improved cloning and detection procedures for the Y2H assay that accelerate the research progress. The changes in procedures involve running an agarose gel after the doubly digested vector and insert are ligated in the cloning step to determine the efficiency of RE digestion and ligation, and performing an additional replica-plating on plates for earlier assessment of interaction in the detection step. We show an example of Y2H interaction between Trs23 and Trs120 (respective subunits of TRAPP I and TRAPP II), as a proof of concept. By following the improved methods described here, the chances of successful cloning increased and the time for the whole Y2H experimental process is significantly shorter.

In Search of Regulators of <i>LeSPL-CNR</i>by South-Western Blotting and Yeast One-Hybrid Library Screening System

LeSPL-CNR is a crucial transcription factor for fruit ripening of Solanum lycopersicum. The cnr (colorless non-ripening) epimutation resulted from hypermethylation in a 286 bp region of LeSPL-CNR promoter inhibits normal fruit ripening. In present study, potential regulators of LeSPL-CNR, which could bind to the specific 286 bp region, were screened via south-western blotting and yeast one-hybrid (Y1H) library screening system. Results indicated that a total of 13 and 19 candidate proteins were acquired respectively, and both ribulose-1,5-bisphosphate carboxylase/oxygenase and 40S ribosomal protein were identified by two methods. These would provide some information for revealing roles of DNA methylation and the regulatory mechanism for LeSPL-CNR.

利用酵母双杂交筛选菠萝AcSWEET11的互作蛋白

SWEET(sugars will eventually be exported transporter)基因在植物开花过程中具有重要的作用,但AcSWEET11在菠萝成花中的作用机制尚不清楚。通过鉴定成花过程中与AcSWEET11的互作蛋白,为菠萝成花机制的解析奠定基础。本研究利用共转化的方法在菠萝成花过程的cDNA膜文库中筛选AcSWEET11的互作蛋白,分析候选蛋白的表达量。结果表明,pBT3-STE-AcSWEET11+pPR3-N对NMY51酵母细胞无毒性,但有自激活活性。进一步研究结果显示,在TDO/3?AT培养基和QDO培养基上自激活受到抑制。利用该系统筛选到了81个阳性克隆,经测序鉴定出48个与AcSWEET11互作的候选蛋白,包括E3 ubiquitin-protein ligase RING1-like、Trehalose-phosphate synthase 7、Cytochrome P450、TranscriptionfactorLUX等。GO和KEGG分析结果显示,48个蛋白主要分布在细胞进程、代谢过程、刺激反应和催化活性等生物过程,参与脂类代谢、氨基酸代谢和碳水化合物代谢、信号转导和运输与分解代谢等新陈代谢途径。Trehalose-phosphate synthase 7(XP_020105459.1)、Protein TIFY 3-like(XP_020082835.1)、40S ribosomal protein S27(XP_020092770.1)、Heterogeneous nuclear ribonucleoprotein 1-like(XP_020112516.1)等4个基因与AcSWEET11表达趋势一致,在菠萝成花过程中下调表达;Dihydrolipoyl dehydrogenase 2(XP_020113798.1)、Putative lipid-transfer protein DIR1(XP_020086640.1)、clathrin assembly protein At4g32285(XP_020108161.1)等3个基因在菠萝成花过程中上调表达。这些结果表明,AcSWEET11可能通过与Trehalose-phosphatesynthase7等蛋白发生互作,参与菠萝成花过程。本研究进一步丰富了AcSWEET11的蛋白互作网络,为AcSWEET11在菠萝成花中的调控机制的解析奠定基础。

长足大竹象信息素结合蛋白CbuqPBP2互作蛋白的筛选与验证

【目的】筛选长足大竹象(Cyrtotrachelus buqueti)信息素结合蛋白CbuqPBP2的互作蛋白,为实现利用信息物质对长足大竹象种群持续控制提供参考。【方法】利用GST pull-down联合质谱技术,对长足大竹象信息素结合蛋白CbuqPBP2的互作蛋白进行筛选、鉴定和分析,并采用酵母双杂交试验验证了CbuqPBP2与信息素结合蛋白CbuqPBP1的特异性互作。【结果】GST pull-down共筛选出45个与长足大竹象信息素结合蛋白CbuqPBP2特异性结合的候选互作蛋白,包括CbuqPBP1、ND2、CYTB。这些互作蛋白主要参与细胞过程、定位、代谢过程、应激反应以及生物调控等多个生物学过程。使用酵母双杂交体系,构建了pGADT7-PBP1重组猎物质粒与pGBKT7-PBP2重组诱饵质粒,通过诱饵质粒毒性检测和自激活检测,表明重组诱饵质粒对Y2HGold酵母菌无毒性作用。共转化验证结果显示,诱饵质粒pGBKT7-PBP2共转化酵母菌株能够在TDO培养基上生长。【结论】CbuqPBP2和CbuqPBP1之间有相互作用,不同信息素结合蛋白间的互作对深入理解长足大竹象嗅觉感受机制提供了新的思路。

橡胶树形成层组织的酵母双杂交cDNA文库构建及HbHDA6互作蛋白筛选

次生乳管是天然橡胶合成和贮存的场所,是由橡胶树树干树皮中维管形成层细胞分裂分化而来。次生乳管的数量与天然橡胶产量直接相关,而这些乳管的数量取决于形成层分化次生乳管的频率(乳管分化能力),是橡胶树产量育种的主要指标。前期研究中,我们发现组蛋白去乙酰化酶(HDA)抑制剂曲古抑菌素A(TSA)能诱导橡胶树乳管分化且组蛋白去乙酰化酶基因(HbHDA6)能够参与橡胶树乳管分化调控。由于组蛋白乙酰化修饰调控橡胶树次生乳管分化的分子机制尚未阐明,因此该文使用冠菌素(COR)诱导橡胶树形成层分化产生次生乳管的实验系统,以分离形成层组织为材料,构建酵母双杂交cDNA文库,以HbHDA6基因为诱饵来筛选酵母双杂交文库,确定与HbHDA6相互作用的蛋白。结果表明:(1)利用Gateway技术构建的均一化COR诱导橡胶树形成层组织的酵母双杂交cDNA文库,初级文库的容量为6.34×10^(6)CFU·mL^(-1),总单克隆数为1.27×10^(7),文库重组率为100%;次级文库的容量为7.72×10^(6)CFU·mL^(-1),总单克隆数为1.54×10^(7),文库重组率为100%。初级文库和次级文库的插入片段平均长度分别为1.1 kb和1.2 kb。(2)成功构建了筛选HbHDA6互作蛋白的pGBKT7-HbHDA6诱饵载体,并确认无自激活活性。(3)使用该诱饵载体对构建的酵母双杂交cDNA文库进行筛选,并通过NCBI_BLAST比对和去除重复以后,获得了22个与HbHDA6发生互作的蛋白,包括CLP1、ERF3、ERF4、HSP82、LARP6a、APT5、PP2A、FBA6等。该研究成果为解析组蛋白乙酰化修饰调控橡胶树次生乳管分化的分子机制提供了理论基础,为转基因改良橡胶树的产胶潜力提供了候选基因,为高性能天然橡胶遗传改良育种提供了新线索。

大豆GmNF-YA13互作蛋白的筛选及鉴定

大豆GmNF-YA13蛋白是一个核转录因子Y(NF-Y),在干旱和高盐响应过程中均发挥重要作用。为研究其抗旱和耐盐的作用机理,寻找GmNF-YA13的互作蛋白,构建pGBKT7-GmNF-YA13诱饵载体,采用酵母双杂交筛选大豆酵母文库,并进行X-α-gal染色验证。结果显示:酵母双杂交获得85个阳性克隆,测序分析后得到36个候选的互作蛋白。功能预测显示互作蛋白主要参与生长发育、胁迫响应、能量代谢、转录调控和信号转导等生物过程。选择GmUVR8、GmCML41、GmFbox13和GmFBA与诱饵pGBKT7-GmNF-YA13进行一对一验证,只有GmFBA能与GmNF-YA13发生相互作用,预示GmNF-YA13功能的发挥需要GmFBA的参与。该结果可为NF-YA抗逆分子网络的研究提供基础。

MsWRKY33转录调控MsACS2影响紫花苜蓿耐盐性的研究

乙烯作为重要的植物激素之一,在植物逆境胁迫应答中发挥重要作用。ACS基因是乙烯合成过程中的关键限速酶(ACC合成酶)基因,本研究通过探究紫花苜蓿MsWRKY33转录因子对ACS基因的调控关系,为MsWRKY33参与紫花苜蓿耐盐胁迫响应的具体调控机制奠定理论基础。利用同源克隆、载体构建、酵母单杂交、酵母双杂交、qRT-PCR等技术,验证MsWRKY33对AtACS2和MsACS2的转录调控,并对盐胁迫下转基因株系中MsACS2表达模式进行分析。结果显示:MsWRKY33转录因子可以与W-box元件特异性结合,且对AtACS2和MsACS2具有转录调控作用;MsWRKY33转基因株系中MsWRKY33基因的表达量显著高于对照,转基因株系中MsACS2的表达是在MsWRKY33基因大量表达后呈上升趋势,进一步说明紫花苜蓿MsACS2基因的表达可能受MsWRKY33转录因子的正向调控。因此,紫花苜蓿受到盐胁迫时可诱导MsWRKY33的表达,MsWRKY33转录因子通过正向调控MsACS2表达,可能影响乙烯合成,从而影响紫花苜蓿的盐胁迫响应。

玉米大斑病菌cDNA文库的构建及转录因子StMR1互作蛋白的筛选

【目的】筛选玉米大斑病菌(Setosphaeria turcica)转录因子的互作蛋白,解析黑色素调控转录因子StMR1调控玉米大斑病菌致病性的分子机制。为解析玉米大斑病菌侵染过程中转录因子的调控网络,阐明病菌的致病机理提供参考。【方法】收集玉米大斑病菌菌丝和孢子不同萌发阶段作为试验材料,采用Gateway方法构建玉米大斑病菌cDNA文库,使用同源重组的方法构建转录因子StMR1的诱饵载体,采用酵母双杂交技术筛选其互作蛋白并进行一对一验证。【结果】构建的玉米大斑病菌文库插入的平均片段长度大于1000 bp,初级文库及次级文库的库容量为1.2×107和1.04×107CFU,重组率为100%,可以用于酵母双杂交筛选。成功构建可以用于筛库的诱饵载体pGBKT7-StMR1,经初筛与复筛得到3个互作蛋白,一对一验证短链脱氢酶、糖基转移酶、富含亮氨酸重复序列蛋白均与转录因子StMR1存在互作。【结论】成功构建了丰富度高且质量好的玉米大斑病菌cDNA文库并筛选到了与转录因子StMR1互作的蛋白。

橡胶树HbPIP2;3的亚细胞定位与多聚化分析

水通道蛋白是一类高效转运水分子的膜内在蛋白,其在生物膜上以四聚体的形式起作用。天然橡胶在橡胶树的乳管细胞中特异合成,并在割胶过程以胶乳的形式被排出。胶乳系乳管细胞的胞质成分,其含水量高达70%,水分通过调节胶乳的粘稠度和乳管膨压进而影响橡胶树的产排胶能力,是决定胶乳产量的关键因素。前期研究显示,橡胶树乳管的水分平衡主要由质膜内在蛋白(plasma membrane intrinsic protein,PIP)特别是HbPIP2;3介导。为揭示HbPIP2;3调控乳管水分平衡的分子机制,本研究采用RT-PCR技术对其861 bp的编码区进行分离。序列分析显示:HbPIP2;3预测编码286个氨基酸,理论分子量为30.58 kDa,等电点为8.50,不稳定系数为31.68,总平均疏水指数为0.449,为稳定的疏水型碱性蛋白;该蛋白含有保守的MIP结构域,其中包括6个典型的跨膜螺旋和2个半螺旋;基于同源建模的3D结构预测显示其可以形成同源四聚体。生物信息学预测和在烟草叶片中的亚细胞定位分析显示,HbPIP2;3定位在细胞膜,这同时也得到了双分子荧光互补(bimolecular fluorescence complementation,BiFC)实验的证实。BiFC实验显示,HbPIP2;3可在细胞膜上形成同源四聚体,这进一步得到酵母双杂交结果的验证。本研究结果表明,HbPIP2;3可通过同源四聚体的方式调控乳管的水分平衡,但是否存在异源互作模式还有待进一步研究。

柱花草SgPAL3基因启动子的克隆及上游转录因子的筛选

柱花草(Stylosanthes spp.)是热带地区广泛种植的重要草种,炭疽病是危害柱花草的严重病害,柱花草SgPAL3基因具有抗炭疽菌的功能。本研究对启动子区域-2 000~0 bp,-1 500~0 bp序列分别构建诱饵载体并检测自激活,结果表明500 ng·mL^(-1)金担子素(Aureobasidin A,AbA)可以抑制两种诱饵载体的自激活。利用Gateway技术构建了柱花草响应炭疽菌的酵母cDNA文库,其容量为1.20×107,插入片段主要分布在750~2 000 bp,重组率为100%;利用酵母单杂交技术,筛选与SgPAL3基因启动子互作的转录因子,并验证了3个转录因子(SgASIL2,SgHAT5和SgZHD8)与SgPAL3启动子的互作关系;qRT-PCR分析表明,SgASIL2,SgHAT5和SgZHD8均响应炭疽菌的侵染,说明它们可能调控柱花草对炭疽病的抗性。本研究为进一步解析SgPAL3响应炭疽菌侵染的转录调控机制奠定了基础。

柱花草SgMPK6互作蛋白的筛选与验证

丝裂原活化蛋白激酶(MAPK)级联反应在调节植物免疫中起关键作用。柱花草炭疽病是危害柱花草生产的严重病害,柱花草SgMPK6基因具有抗炭疽菌的功能。为探究响应胶孢炭疽菌侵染的柱花草SgMPK6下游互作蛋白,本研究采用酵母双杂交技术,以SgMPK6激酶结构域作为诱饵蛋白,筛选柱花草cDNA文库,共获得74个与SgMPK6激酶结构域潜在的互作蛋白,并通过酵母双杂交点对点试验,验证了候选互作蛋白SgbHLH32、SgbHLH33、SgbHLH44与SgMPK6激酶结构域间的互作关系。磷酸化位点预测显示,3个bHLH转录因子均具有MAPKs潜在的磷酸化位点。柱花草响应炭疽菌侵染的qRT-PCR分析表明,SgbHLH32、SgbHLH33、SgbHLH44转录因子均显著上调,预示互作蛋白可能作为SgMPK6的底物调控柱花草对炭疽病的抗性。本研究为进一步解析SgMPK6响应炭疽菌侵染的分子机制提供了试验依据。