Analyzing the homeostasis of signaling proteins by a combination of Western blot and fluorescence correlation spectroscopy

The determination of intracellular protein concentrations is a prerequisite for understanding protein interaction networks in systems biology. Today, protein quantification is based either on mass spectrometry, which requires large cell numbers and sophisticated measurement protocols, or on quantitative Western blotting, which requires the expression and purification of a recombinant protein as a reference. Here, we present a method that uses a transiently expressed fluorescent fusion protein of the protein-of-interest as an easily accessible reference in small volumes of crude cell lysates. The concentration of the fusion protein is determined by fluorescence correlation spectroscopy, and this concentration is used to calibrate the intensity of bands on a Western blot. We applied this method to address cellular protein homeostasis by determining the concentrations of the plasma membrane-located transmembrane scaffolding protein LAT and soluble signaling proteins in naïve T cells and transformed T-cell lymphoma (Jurkat) cells (with the latter having nine times the volume of the former). Strikingly, the protein numbers of soluble proteins scaled with the cell volume, whereas that of the transmembrane protein LAT scaled with the membrane surface. This leads to significantly different stoichiometries of signaling proteins in transformed and naïve cells in concentration ranges that may translate directly into differences in complex formation.     

An efficient protein complex purification method for functional proteomics in higher eukaryotes

The ensemble of expressed proteins in a given cell is organized in multiprotein complexes. The identification of the individual components of these complexes is essential for their functional characterization. The introduction of the 'tandem affinity purification' (TAP) methodology substantially improved the purification and systematic genome-wide characterization of protein complexes in yeast. The use of this approach in higher eukaryotic cells has lagged behind its use in yeast because the tagged proteins are normally expressed in the presence of the untagged endogenous version, which may compete for incorporation into multiprotein complexes. Here we describe a strategy in which the TAP approach is combined with double-stranded RNA interference (RNAi) to avoid competition from corresponding endogenous proteins while isolating and characterizing protein complexes from higher eukaryotic cells. This strategy allows the determination of the functionality of the tagged protein and increases the specificity and the efficiency of the purification.     

In situ chemical cross-linking on living cells reveals CD9P-1 cis-oligomer at cell surface

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. They associate with each other in multimolecular complexes containing numerous membrane proteins. As a first step towards the study of the supramolecular organization of tetraspanin complexes, we have implemented a proteomic approach based on in situ protein cross-linking on living cells followed by affinity purification of tetraspanin complexes. This allowed observing the presence of high molecular weight protein complexes that were characterized as containing CD9P-1/CD315 using LC-MS/MS. Western blot analyses and the use of different tags demonstrated the presence of CD9P-1 oligomer in cis-association at cell surface. A significant amount of CD9P-1 oligomer was observed on various cell types. We have shown that CD9P-1 self-associates independently from its association with tetraspanins. However, the expression level of CD9 or CD81 that associate directly and specifically with CD9P-1, positively modulates the cross-linking efficiency of CD9P-1. Thus, tetraspanins can play a role on CD9P-1 oligomerization status.     

In situ chemical cross-linking on living cells reveals CD9P-1 cis-oligomer at cell surface

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. They associate with each other in multimolecular complexes containing numerous membrane proteins. As a first step towards the study of the supramolecular organization of tetraspanin complexes, we have implemented a proteomic approach based on in situ protein cross-linking on living cells followed by affinity purification of tetraspanin complexes. This allowed observing the presence of high molecular weight protein complexes that were characterized as containing CD9P-1/CD315 using LC-MS/MS. Western blot analyses and the use of different tags demonstrated the presence of CD9P-1 oligomer in cis-association at cell surface. A significant amount of CD9P-1 oligomer was observed on various cell types. We have shown that CD9P-1 self-associates independently from its association with tetraspanins. However, the expression level of CD9 or CD81 that associate directly and specifically with CD9P-1, positively modulates the cross-linking efficiency of CD9P-1. Thus, tetraspanins can play a role on CD9P-1 oligomerization status.     

快速纯化重组腺联病毒的受体结合捕捉方法

【目的】建立用于重组腺联病毒(AAV)纯化的受体结合捕捉方法。【方法】将AAV受体的多囊肾病(PKD)结构域1和2与类弹性蛋白多肽(ELP)在重组大肠杆菌中进行融合表达,利用相变循环(ITC)纯化ELP-PKD融合蛋白;分别用昆虫和AAV-293细胞制备rAAV-GFP,与ELP-PKD融合蛋白共孵育后进行ITC,从沉淀复合物提取病毒DNA进行PCR检测;在优化条件下利用ELP-PKD蛋白结合捕捉rAAV-GFP,利用电子显微镜观察、免疫转印和细胞感染试验进行rAAV鉴定。【结果】ELP-PKD融合蛋白获得正确、可溶性表达,ITC纯化的蛋白纯度大于90%;ELP-PKD蛋白能特异结合rAAV-GFP,结合具有p H、温度和时间依赖性,受体结合捕捉方法可在1h内完成,从两种细胞纯化rAAV-GFP的回收率分别为58%和56%;rAAV-GFP洗脱具有p H和温度依赖性,洗脱rAAV-GFP的回收率分别为46%和44%;纯化rAAV-GFP具有AAV的典型形态和结构蛋白。【结论】建立的ELP-PKD结合捕捉法可用于不同细胞源rAAV的快速纯化。     

Ions of the interactome: the role of MS in the study of protein interactions in proteomics and structural biology

The role of MS in the study of protein-protein interactions in solution is described from a proteomics perspective, in terms of high-throughput analyses of protein complexes in vivo, through to chemical and biochemical treatments ahead of MS analysis in the context of complementary experimental approaches in structural biology. The use of MS to characterise protein-protein interactions is described following the single and tandem affinity purification of protein complexes and assemblies of expressed proteins in host cells, the isolation and preservation of protein complexes on surfaces and microarrays, and their prior treatment with chemical and biochemical probes by hydrogen exchange, radical probe, chemical cross-linking, and limited proteolysis. The advantages and disadvantages of each of the approaches are presented. These new and emerging applications, which further demonstrate the power of MS, continue to ensure that the mass spectrometer will remain at the heart of discoveries in proteomics in the foreseeable future.     

Isolation of viral ribonucleoprotein complexes from infected cells by tandem affinity purification

The biochemical purification and analysis of viral ribonucleoprotein complexes (RNPs) of negative-strand RNA viruses is hampered by the lack of suitable tags that facilitate specific enrichment of these complexes. We therefore tested whether fusion of the tandem-affinity-purification (TAP) tag to the main component of viral RNPs, the nucleoprotein, might allow the isolation of these RNPs from cells. We constitutively expressed TAP-tagged nucleoprotein of Borna disease virus (BDV) in cells persistently infected with this virus. The TAP-tagged bait was efficiently incorporated into viral RNPs, did not interfere with BDV replication and was also packaged into viral particles. Native purification of the tagged protein complexes from BDV-infected cells by two consecutive affinity columns resulted in the isolation of several viral proteins, which were identified by MS analysis as the matrix protein, the two forms of the nucleoprotein and the phosphoprotein. In addition to the viral proteins, RT-PCR analysis revealed the presence of viral genomic RNA. Introduction of further protease cleavage sites within the TAP-tag significantly increased the purification yield. These results demonstrate that purification of TAP-tagged viral RNPs is possible and efficient, and may therefore provide new avenues for biochemical and functional studies of these complexes.     

水稻Qb-SNARE蛋白OsNPSN11多克隆抗体制备、鉴定与应用

在真核生物细胞囊泡运输过程中的膜融合主要是由SNARE蛋白介导的, OsNPSN11是从水稻中克隆的Qb-SNARE家族基因, 文章将OsNPSN11构建到原核表达载体pET-30a中与6个His标签融合, 重组质粒pET-OsNPSN11转化BL21(DE3)0.5 mmol/L IPTG诱导4 h后获得了高效表达。用镍离子亲和树脂(Ni²⁺-NTA His Bind Resin) 纯化融合蛋白, 以纯化后的蛋白为抗原免疫新西兰家兔制备多克隆抗体, Western blotting结果显示, 该抗体能特异识别在原核系统表达的抗原, 以及水稻不同组织质膜组分中的OsNPSN11, 可用于转基因水稻中目标蛋白的表达分析。     

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners.     

表达GP64的重组HaSNPV的构建研究

杆状病毒的出芽病毒具有两类不同的膜融合蛋白, 组 I类型的杆状病毒利用的是膜融合蛋白GP64, 而组 II类型的杆状病毒利用的是膜融合蛋白F。本文以组II类型的HaSNPV为研究对象, 研究组I类病毒的GP64能否在组 II类病毒中正确表达和包装。利用 Bac to Bac系统, 构建了带有 AcMNPV膜融合蛋白 GP64 和增强型绿色荧光蛋白的重组病毒HaSNPVgp64 egfp , 同时构建了仅带有增强型绿色荧光蛋白的对照重组病毒 HaSNPVegfp , 通过对HaSNPVgp64 egfp 感染的HzAM1细胞及所产生的子代BV的Western blot检测, 证明GP64可在HzAM1中表达, 并被包装入子代BV。     

Comparison of strong cation exchange and SDS-PAGE fractionation for analysis of multiprotein complexes

Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. Although affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.     

Eukaryotic integral membrane protein expression utilizing the <Emphasis Type="Italic">Escherichia coli</Emphasis> glycerol-conducting channel protein (GlpF)

A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous membrane-protein expression is demonstrated by the expression and insertion into the E. coli cell membrane of the human membrane proteins: occludin, claudin 4, duodenal ferric reductase and a J-type inwardly rectifying potassium channel. The proteins are produced with C-terminal hexahistidine tags (to permit purification of the expressed fusion proteins using immobilized metal affinity chromatography) and a peptidase cleavage site (to allow recovery of the unfused eukaryotic protein).     

Fluorescent proteins as proteomic probes

Protein complexes mediate the majority of cellular processes. Knowledge of the localization and composition of such complexes provides key insights into their functions. Although green fluorescent protein (GFP) has been widely applied for in vivo visualization of proteins, it has been relatively little used as a tool for the isolation of protein complexes. Here we describe the use of the standard GFP tag to both visualize proteins in living cells and capture their interactions via a simple immunoaffinity purification procedure. We applied this method to the analysis of a variety of endogenous protein complexes from different eukaryotic cells. We show that efficient isolations can be achieved in 5-60 min. This rapid purification helps preserve protein complexes close to their original state in the cell and minimizes nonspecific interactions. Given the wide use and availability of GFP-tagged protein reagents, the present method should greatly facilitate the elucidation of many cellular processes.     

牛病毒性腹泻病毒E^rns-E2融合蛋白在果蝇S2细胞中的表达与鉴定

目的：利用果蝇S2细胞表达牛病毒性腹泻病毒（BVDV）Erns-E2融合蛋白,并对其抗体结合能力进行鉴定。方法：用RT-PCR方法扩增BVDV NADL株Erns和E2蛋白的编码基因,利用（G4-S）3柔性15肽基因将扩增的2个基因连接,再与昆虫表达载体pMT/BiP/V5-His连接构建重组表达载体pMT/BiP/V5-His-Erns-E2,将后者与筛选质粒pCoBlast共转染果蝇S2细胞后表达Erns-E2融合蛋白,并对表达产物进行鉴定。结果：SDS-PAGE结果表明,融合蛋白相对分子质量为76800;Western blotting检测表明,该融合蛋白具有与BVDV抗体良好的结合能力。结论：BVDV的Erns-E2融合蛋白能在果蝇S2细胞中进行表达;经鉴定,表达产物具有良好的抗体结合能力,可用于抗原检测。     

口蹄疫病毒P1基因在大肠杆菌中的高效表达及其生物活性的初步分析

将口蹄疫病毒 (FMDV)结构蛋白基因P1的完整cDNA序列插入原核表达性载体pGEX KG中 ,使P1基因与GST融合 ,获得融合表达质粒pKG P1,转化E .coliBL₂₁ (DE3) ,经IPTG诱导 ,SDS PADE结果表明GST P1融合蛋白获得高效表达 ,Western blot检测证实表达的融合蛋白具有免疫学活性 ,表达产物主要存在于细菌裂解液上清中。进一步采用GST纯化试剂盒纯化P1蛋白并作为诊断抗原 ,建立了P1 ELISA诊断方法 ,与FMD间接血凝 (IHA)检测方法平行检测 86 4份血清样品 ,总的符合率达87%。     

Identification of different isoforms of eEF1A in the nuclear fraction of human T-lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers.

GT oligomers, showing a dose-dependent cytotoxic effect on a variety of human cancer cell lines, but not on normal human lymphocytes, recognize and form complexes with nuclear proteins. By working with human T-lymphoblastic CCRF-CEM cells and by using MS and SouthWestern blotting, we identified eukaryotic elongation factor 1 alpha (eEF1A) as the main nuclear protein that specifically recognizes these oligonucleotides. Western blotting and supershift assays confirmed the nature of this protein and its involvement in forming a cytotoxicity-related complex (CRC). On the contrary, normal human lymphocytes did not show nuclear proteins able to produce CRC in a SouthWestern blot. Comparative bidimensional PAGE and Western-blotting analysis for eEF1A revealed the presence of a specific cluster of spots, focusing at more basic pH, in nuclear extracts of cancer cells but absent in those of normal lymphocytes. Moreover, a bidimensional PAGE SouthWestern blot demonstrated that cytotoxic GT oligomers selectively recognized the more basic eEF1A isoform expressed only in cancer cells. These results suggest the involvement of eEF1A, associated with the nuclear-enriched fraction, in the growth and maintenance of tumour cells, possibly modulated by post-translational processing of the polypeptide chain.     

小鼠β-防御素30的原核表达和纯化

目的:在原核细胞中表达小鼠β-防御素30(DEFB30),并对表达产物进行鉴定和纯化。方法:用RT-PCR方法扩增小鼠Defb30的cDNA序列,将2个拷贝的cDNA序列串联连入原核表达载体pET28(a),构建重组表达载体pET28(a)-Defb30,并将重组表达载体转化至大肠杆菌Rosetta(DE3),IPTG诱导表达,以Western印迹分析表达产物His-DEFB30,用Ni-NTA亲和柱纯化融合蛋白。结果:构建了Defb30基因的原核表达载体,经IPTG诱导,相对分子质量约15×103的融合蛋白获得表达,Western印迹分析证实此蛋白即为目的蛋白,经Ni-NTA柱亲和纯化,获得了高纯度的融合蛋白His-DEFB30。结论:获得了在大肠杆菌中表达的DEFB30,为研究该蛋白的免疫避孕效果、抗菌活性奠定了基础。     

Nesprin2在小鼠睾丸各级生精细胞中的表达及其原核表达与纯化

目的:研究Nesprin2在小鼠睾丸各级生精细胞中的表达变化,并对Nesprin2进行原核表达和纯化,为进一步研究该蛋白在精子发生中的作用奠定基础。方法:制备小鼠睾丸细胞涂片,用Nesprin2特异性抗体进行细胞免疫荧光染色,观察Nesprin2在各级生精细胞中的表达;应用RT-PCR技术扩增Nesprin2 C端包含KASH domain的编码85个氨基酸的目的片段,将PCR产物插入到PUCm-T载体中测序,将测序验证后的目的片段亚克隆至原核表达载体PGEX-4T-1中,转化大肠杆菌BL21,IPTG诱导表达。对GST-Nesprin2 C85融合蛋白进行纯化,Western blot进行验证。结果:免疫荧光检测结果发现,Nesprin2在小鼠睾丸各级生精细胞中均有表达,主要分布在核膜及其周围,在减数分裂过程中Nesprin2可能参与核膜重塑。构建了PGEX-4T-1-Nesprin2 C85重组质粒,经IPTG诱导后成功表达了GST-Nesprin2 C85融合蛋白。对GST-Nesprin2 C85融合蛋白进行纯化后,Western blot进行检测,结果发现,原核诱导表达的融合蛋白为GST-Nesprin2 C85融合蛋白。结论:Nesprin2在小鼠各级生精细胞中均有表达,在减数分裂过程中可能参与核膜的重塑。     

Purification of Porcine Phospholamban Expressed inEscherichia coli

Phospholamban (PLB) is a small hydrophobic protein that regulates contractility in the heart. This membrane protein expressed in bacterial cells is resistant to purification by conventional strategies that have been used to isolate expressed soluble proteins. Therefore, in order to obtain both wild-type and mutant PLB proteins, we have amplified the PLB gene by the polymerase chain reaction from genomic DNA of porcine heart and inserted it into the pGEX-2T plasmid expression vector. In this vector, the gene product fused to glutathioneS-transferase has been expressed in JM109Escherichia colicells. The expressed fusion protein was found associated predominantly with insoluble cellular constituents. However, most of the fusion protein was readily extracted with SDS. PLB was subsequently purified by a simple procedure consisting of isolation of the fusion protein by preparative SDS–gel electrophoresis, followed by a second electrophoretic separation of PLB after its cleavage from the fusion protein by thrombin. This isolation method yields 3–4 mg of PLB per liter of cells, in a form which is capable of functional interaction with the Ca-ATPase in reconstituted proteoliposomes.     

Expression of non-structural protein NS3 gene of Bombyx mori densovirus （China isolate）

The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 (China isolate),named BmDNV-3,is a kind of bidensovirus.It is a new type of virus with unique replication mechanisms.To investigate the effects of the NS3 gene during viral DNA replication,a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus (China isolate).Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe,respectively.The NS3 protein was expressed in Escherichia coli BL21.The pFastBacHTe-NS3 was transformed to E.coli DH10Bac.The recombinant bacmid baculoviruses (rBacmid-EGFP-NS3)isolated from the white colonies were transfected into BraN-4 cells using a transfection reagent.BmN-4 cells were infected with recom-binant virus to express fusion proteins.The expression of fusion protein around 30 kDa in E.coli BL21 was identified by SDS-PAGE,Western blotting,and mass spectrometry.The expressed NS3 protein by B.mor/nucleopolyhedrovirus bacmid system was confirmed byWestern blotting using an anti-NS3 polyclonal antibody.And about 45 kDa protein was found.The expressed fusion protein was smalleithan the expected size of EGFP-NS3,55 kDa.Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected lar-vae with smaller molecular size.