基于人类蛋白质图集的蛋白质表达的总论

新的人类蛋白质图集4.0版本上已经含有了对应5000个人类基因的6000多种抗体。这个版本里已经拥有500多万张高分辨率的免疫组化和激光共聚焦图片。每张图片都是经过优秀的病理学家的注释,从而为功能研究提供知识储备,也可以进行正常和病理组织中蛋白质表达谱的查询和文献检索。一个新的结构实现了,它包括了所有预测的基因(大约20400个),并且带有可视化的所有编码蛋白质基因的特征。一个新的搜索工具也已经启动了,它可以执行高级检索功能,包括染色体定位、蛋白质分级和(或)组织特异性的检索。蛋白质图集作为一种搜索工具可以发现癌症诊断学的潜在生物标志物。     

Production of H-Y antibody in the ascites fluid of mouse and localization of the antigen on cells and tissues

A procedure is described for the production of large amounts of ascites fluid containing specific H-Y antibody. The distribution of H-Y antigen on mouse epididymal spermatozoa, thymocytes, and splenocytes was carried out using this specific antibody in the microcytotoxicity test and ELISA. Employing the indirect immunofluorescent technique, the H-Y antigen was localized on the acrosomal membrane of mouse epididymal and washed ejaculated human spermatozoa and on the entire membrane of mouse splenocytes and thymocytes. Immunohistochemical localization of the antigen in the testicular section indicated its presence in the cytoplasm of Leydig cells and on the membrane of Sertoli cells and sperm heads.     

WWOX protein expression in normal human tissues

WWOX is a putative tumor suppressor gene that spans approximately a 1 Mb genomic region and is the site for the second most common chromosomal fragile site, FRA16D at 16q23. Various studies have focused on the expression of WWOX in human cancer mostly at the RNA level, but little is known about the normal pattern of WWOX protein expression in non-neoplastic tissues. In this study, a comprehensive analysis of WWOX protein expression in normal tissues was performed by means of immunohistochemistry utilizing a very specific anti-WWOX polyclonal antibody. We analyzed tissue cores of human samples representing more than 30 organs, using various tissue microarray (TMA) slides. Due to the potential role of WWOX in sex-steroid metabolism, whole sections from hormonally regulated organs like breast, ovaries, testes and prostate were also analyzed. The results from our study indicate that WWOX is preferentially highly expressed in secretory epithelial cells of reproductive, endocrine and exocrine organs, as well as in ductal epithelial cells from specific segments of the urinary system. Interestingly, we also observed significant WWOX protein expression in various cell types of neural origin including neurons, ependymal cells and astrocytes. No expression of WWOX was detected in adipose, connective, and lymphoid tissues, myelinized structures and blood vessels. By better defining the topographic distribution of WWOX in normal tissues this study provides some insight on the potential physiological role of this novel protein.     

Distribution of obestatin and ghrelin in human tissues: immunoreactive cells in the gastrointestinal tract, pancreas, and mammary glands.

Obestatin and ghrelin are two peptides derived from the same prohormone. It is well established that ghrelin is produced by endocrine cells in the gastric mucosa. However, the distribution of human obestatin immunoreactive cells is not thoroughly characterized. A polyclonal antibody that specifically recognizes human obestatin was produced. Using this antibody and a commercial antibody vs ghrelin, the distribution of obestatin and ghrelin immunoreactive cells was determined in a panel of human tissues using immunohistochemistry. The two peptides were detected in the mucosa of the gastrointestinal tract, from cardia to ileum, and in the pancreatic islets. Interestingly, epithelial cells in the ducts of mammary glands showed distinct immunoreactivity for both ghrelin and obestatin. By double immunofluorescence microscopy, it was shown that all detected cells were immunoreactive for both peptides. Furthermore, the subcellular localization of obestatin and ghrelin was essentially identical, indicating that obestatin and ghrelin are stored in the same secretory vesicles.     

The antibody preparation and expression of human Pescadillo

To explore the biological roles of human Pescadillo and investigate its potential effect on tumorigene sis, the eDNA of Pescadillo was fused with that of GST. After purification and elution, the purified GST-Pescadillo fusion protein was obtained, and the antibody against the fusion protein was generated.Endogenous Pescadillo protein was observed to be remarkably induced by estrogen. It was mainly distributed in the tissues such as breast, ovary and intestine, all of which contain proliferating cells,and was also detected in many cell lines of human cancer: renal carcinoma, hepatoma, ovarian cancer,colon carcinoma, and breast cancer. The expression level of Pescadillo was increased significantly in breast cancer tissues compared with their paired margin tissues. Taken together, these data suggest that Pescadillo may play important roles in the initiation and development of cancer and may be a potential target in cancer diagnosis and therapy.     

Constitutive expression and cytoplasmic compartmentalization of ATM protein in differentiated human neuron-like SH-SY5Y cells

Ataxia telangiectasia (A-T) is an autosomal, recessive disorder mainly characterized by neuronal degeneration. However, the reason for neuronal degeneration in A-T patients is still unclear. ATM (A-T, mutated), the gene mutated in A-T, encodes a 370-kDa protein kinase. We measured the levels of the ATM protein found in differentiated neuron-like rat PC12 cells and differentiated neuron-like human SH-SY5Y cells. We found that, in rat PC12 cells, ATM levels decreased dramatically after differentiation, which is consistent with previous results observed in differentiated mouse neural progenitor cells. In contrast, the levels of ATM were similar before and after differentiation in human SH-SY5Y cells. Using an indirect immunofluorescence assay, we showed that ATM translocates from the nucleus to the cytoplasm in differentiated human SH-SY5Y cells. The translocation of ATM was further verified by subcellular fractionation experiments. The constitutive expression and cytoplasmic translocation of ATM in differentiated SH-SY5Y cells suggest that ATM is important for maintaining the regular function of human neuronal cells. Our results further demonstrated that, in response to insulin, ATM protects differentiated neuron-like SH-SY5Y cells from serum starvation-induced apoptosis. These data provide the first evidence that cytoplasmic ATM promotes survival of human neuronal cells in an insulin-dependent manner.     

High-level expression, polyclonal antibody preparation and sub-cellular localization analysis of mouse Rhox5 protein

Mouse reproductive homeobox on the X chromosome (Rhox) is a novel homeobox gene cluster. Rhox5, also called Pem, belongs to the beta subcluster of Rhox. Codon analysis indicated that the cDNA contains 16% of codons rarely used in Escherichia coli. To achieve high-level expression of Rhox5, the coding sequence of Rhox5 was amplified and subcloned into the prokaryotic expression vector pET22b (+) in order to produce 6His-tagged fusion protein in the modified BL21 (DE3) cells, namely Rosetta2 (DE3) cells. The 6His-tagged Rhox5 was expressed efficiently in Rosetta2 (DE3), compared with marginal expression in BL21 (DE3). The fusion protein amounted to 16% of the total bacterial proteins after induction with 0.4mM IPTG for 1.5h at 37 degrees C. After purification, Rhox5-6His was used to immunize New Zealand white rabbits following standard protocol. The homemade antiserum could detect both endogenous Rhox5 protein expressed in eukaryotic cells (Cos-7) and exogenous GFP-Rhox5 protein. Furthermore, the antiserum was used to determine the localization of Rhox5 in NIH3T3 cells using an immunofluorescence technique. The results demonstrated that Rhox5 was localized predominantly in the nucleus. Preparation of the anti-Rhox5 polyclonal antibody will facilitate further functional study of Rhox5.     

A 205 kDa protein from non-neuronal cells in culture contains tubulin binding epitopes

Microtubule-associated proteins (MAPs) interact with tubulinin vitro andin vivo. Despite that there is a large amount of information on the roles of these proteins in neurons, the data on non-neuronal MAPs or MAPs-related proteins is scarce. There is an increasing number of microtubule-interacting proteins that have been identified in different cultured cell lines, and some of them share common functional epitopes with the most well-known MAPs, MAP-2 and tau. In a search for tubulin-interacting proteins in non-neuronal cells we identified a 205 kDa protein in the monkey kidney Vero cells in culture, on the basis of immunological studies and affinity chromatography. This protein interacts with the C-terminal moiety of -tubulin and cosediments with taxol assembled microtubules, but it was not recovered after successive cycles of assembly and disassembly. The presence of neuronal MAPs such as MAP-1, MAP-2 and tau was not detected in these cells. Interestingly, the studies showed that the 205 kDa protein contained a tubulin binding motif which was recognized by site-directed antibodies that also tag tubulin binding epitopes on MAP-2 and tau. This characteristic led us to designate this protein as MBD-205, a component that shares binding domains with these MAPs, rather than as a marker of the MAPs family. On the other hand, immunofluorescence experiments using site-specific antibodies, i.e. MAP-reacting monoclonal anti-idiotypic reagent MTB6.22 and a polyclonal antibody to the second tau repeat, revealed a MBD-205 co-localization with membrane structures and microtubule-organizing centers in Vero cells. Microinjection studies along with studies on the cell distribution suggest that MBD-205 appears to play a structural role at the level of the microtubule interactions in these cells.     

Expression of calcyclin-binding protein/Siah-1 interacting protein in normal and malignant human tissues: an immunohistochemical survey.

Calcyclin-binding protein (CacyBP)/Siah-1 interacting protein (SIP), a component of ubiquitin-mediated proteolysis, could bind the Skp1-Cul1-F box protein complex. Although CacyBP/SIP was implicated in p53-induced beta-catenin degradation, its exact function was still unknown. Our previous studies showed that CacyBP/SIP could modulate the multidrug-resistant phenotype of gastric cancer cells and was highly expressed in gastric cancer tissues compared with that in non-cancerous tissues. In this study, CacyBP/SIP protein expression profile in a broad range of human normal tissues and carcinomas was analyzed by immunohistochemistry staining with anti-CacyBP/SIP monoclonal antibody first produced in our laboratory. CacyBP/SIP was generally localized in the cytoplasm/nucleus. Positive staining of CacyBP/SIP was found in brain, heart, lymph node, and esophagus. Weak staining was shown in the rectum and kidney. No CacyBP/SIP was detected in other normal tissues. However, CacyBP/SIP was ubiquitously detected in all kinds of tumor tissues and was highly expressed in nasopharyngeal carcinoma, osteogenic sarcoma, and pancreatic cancer. To our knowledge, this is the first study on the CacyBP/SIP expression pattern in a broad range of human normal and tumor tissues. The data presented should serve as a useful reference for other investigators in future studies of CacyBP/SIP functions. Hopefully, this knowledge will lead to discovery of more roles of CacyBP/SIP in tumorigenesis.     

gC1qR expression in normal and pathologic human tissues: differential expression in tissues of epithelial and mesenchymal origin

The gC1qR (i.e., gC1q receptor, gC1q binding protein, p32, p33) is a multifunctional cellular protein that interacts with components of the complement, kinin, and coagulation cascades and select microbial pathogens. Enhanced gC1qR expression has been reported in adenocarcinomas arising in a variety of organs. The present study compared gC1qR expression in normal, inflammatory, dysplastic, and malignant tissue of epithelial and mesenchymal origin. gC1qR expression was visualized in tissue sections by immunohistochemistry using the 60.11 monoclonal antibody (i.e., IgG(1) mouse monoclonal antibody directed against gC1qR) and the UltraVision LP Detection System. Sections were counterstained with hematoxylin and examined by light microscopy. Strongest gC1qR expression was noted in epithelial tumors of breast, prostate, liver, lung, and colon, as well as in squamous and basal cell carcinoma of the skin. However, increased gC1qR staining was appreciated also in inflammatory and proliferative lesions of the same cell types, as well as in normal continuously dividing cells. In contrast, tumors of mesenchymal origin generally stained weakly, with the exception of osteoblasts, which stained in both benign and malignant tissues. The data suggest that increased gC1qR expression may be a marker of benign and pathologic cell proliferation, particularly in cells of epithelial origin, with potential diagnostic and therapeutic applications.     

Effects of increased cellular zinc levels on gene and protein expression in HT-29 cells

High cellular zinc concentrations lead to impairments in ATP synthesis and cell cycle control particularly in neurons and epithelial cells. The molecular basis for these dysfunctions is still not fully elucidated. Here we analyzed the effects of a high zinc exposure (10ppm) on gene and protein expression in the human epithelial cell line HT-29. Of the 1176 genes analyzed with cDNA arrays, nine differentially expressed genes were identified. Proteome analysis based on 1310 detected proteins identified 11 molecular targets. Most of the identified genes/proteins have not been linked to cellular zinc status before (e.g. PEC-60, R-ras3). More than half of the targets participate in ATP production or stress response. Therefore, it appears that higher zinc concentrations mediate their effects mainly via impairments in cellular energy metabolism and stress response.This work was in part supported by the Degussa Bio Actives GmbH, Freising-Weihenstephan, Germany.     

Chimeric prion protein expression in cultured cells and transgenic mice.

The efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558-1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the functionality of particular regions of the PrP molecule. Previous reports demonstrated that the PrP gene specifies the host range for susceptibility of transgenic animals to prions (Scott, M., et al., 1989, Cell 59, 847-857; Prusiner, S.B., et al., 1990, Cell 63, 673-686). Consistent with these results, we showed that Syrian hamster (SHa) PrP is ineligible for efficient conversion to PrPSc in ScN2a cells. By constructing a series of chimeric mouse (Mo)/SHaPrP genes, we developed an epitopically tagged functional variant of the MoPrP gene, which can efficiently form protease-resistant PrP molecules upon expression in ScN2a cells. The presence of a defined epitope for an SHa-specific monoclonal antibody allows the products of this chimeric gene to be discriminated from endogenous MoPrP and creates a useful reagent for exploring structure/function relationships via targeted mutagenesis. In addition, we developed a transgenic mouse expression vector by manipulation of an SHaPrP cosmid clone. This vector permits the efficient expression of foreign PrP genes in the brains of transgenic animals, enabling pathological consequences of in vitro mutagenesis to be studied.     

Constitutive expression of human pancreatic lipase-related protein 1 in Pichia pastoris

High-level constitutive expression of the human pancreatic lipase-related protein 1 (HPLRP1) was achieved using the methylotrophic yeast Pichia pastoris. The HPLRP1 cDNA, including its original leader sequence, was subcloned into the pGAPZB vector and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. A major protein with a molecular mass of 50 kDa was found to be secreted into the culture medium and was identified using anti-HPLRP1 polyclonal antibodies as HPLRP1 recombinant protein. The level of expression reached 100-120 mg of HPLRP1 per liter of culture medium after 40 h, as attested by specific and quantitative enzyme-linked immunosorbent assay. A single cation-exchange chromatography sufficed to obtain a highly purified recombinant HPLRP1 after direct batch adsorption onto S-Sepharose of the HPLRP1 present in the culture medium, at pH 5.5. N-terminal sequencing and mass spectrometry analysis were carried out to monitor the production of the mature protein and to confirm that its signal peptide was properly processed.     

The soluble expression of the human renin binding protein using fusion partners: A comparison of ubiquitin,thioredoxin, maltose binding protein and NusA

human renin binding protein (hRnBp), showingN-acetylglucosamine-2-epimerase activity, was over-expressed inE. coli, but was mainly present as an inclusion body. To improve its solubility and activity, ubiquitin (Ub), thioredoxin (Trx), maltose binding protein (MBP) and NusA, were used as fusion partners. The comparative solubilities of the fusion proteins were, from most to least soluble: NusA, MBP, Trx, Ub. Only the MBP fusion did not significantly reduce the activity of hRnBp, but enhanced the stability. The Origami (DE3), permitting a more oxidative environment for the cytoplasm inE. coli, helped to increase its functional activity.     

A balanced expression of two chains of heterodimer protein, the human interleukin-12, improves high-level expression of the protein in CHO cells

Interleukin-12 (IL-12) comprises of p40 and p35 subunits that are encoded by genes on separate chromosomes and form p70 heterodimer, a bioactive protein, and free p40, an antagonist of IL-12. Balance expression of two subunits within cells would be the key for high-level of production of bioactive IL-12. Thinking about different expression efficiencies of two genes (p40 gene with higher efficiency), we selected two expression vectors with different efficiencies and inserted genes of p40 and p35 into them separately and co-transfected them into Chinese hamster ovary (CHO) cells. The high-level expression of IL-12 was obtained when p40 cDNA was inserted into pcDNA3 (lower expression vector) and p35 cDNA was inserted into pEE14 (higher expression vector), but using pEE14 for p40 cDNA and pcDNA3 for p35 cDNA, which was opposite to the optimal design, or pEE14 or pcDNA3 for both p35 cDNA and p40 cDNA did not obtain high-level of production of p70 heterodimer, the bioactive IL-12. We also observed that using two chemical reagents in combination, as a pressure selection method or amplification for the two vectors, markedly enhanced the IL-12 production, when compared with any one selection chemical. Our results indicated that the balance expressions of two chains of hetrodimer protein, such as p40 and p35 of IL-12, would be a better choice to obtain high-level of production of the proteins.     

Broad immunocytochemical localization of the formylpeptide receptor in human organs, tissues, and cells

The formylpeptide receptor (FPR), previously found only on polymorphonuclear leukocytes and monocytes/macrophages, responds to both synthetic N-formyl oligopeptides and those produced by bacteria. The cDNA for human FPR has been cloned and a rabbit polyclonal antiserum directed against a synthetic 11-amino-acid peptide corresponding to the deduced carboxy-terminus has been produced. We have now extensively characterized and used the antibody to detect FPR on normal human tissues and cell types. The receptor antigen is present on some epithelial cells, especially those with a secretory function, and on some endocrine cells, e.g., follicular cells of the thyroid and cortical cells of the adrenal. Liver hepatocytes and Kupffer cells are positive. Smooth muscle and endothelial cells are also generally positive. In the brain and spinal cord, the neurons of the motor, sensory, and cerebellar systems, and those of the parasympathetic and sympathetic systems stain positively. These data suggest that the putative endogenous agonist for FPR or an antigenically similar receptor reacts with cellular targets in the neuromuscular, vascular, endocrine, and immune systems.     

A first global analysis of plasmid encoded proteins in the ACLAME database

Many plasmids are mobile genetic elements (MGEs) and, as other members of that group of DNA entities, their genomes display a mosaic and combinatorial structure, making their classification extremely difficult. As other MGEs, plasmids play a major role in horizontal transfer of genetic materials and genome reorganization. Yet, the full impact of such phenomenon on major properties of the host cell, such as pathogenicity, the ability to use new carbon sources or resistance to antibiotics, remains to be fully assessed. More and more complete plasmid genome sequences are available. However, in the absence of standards for storing plasmid sequence data and annotating genes and gene products on sequenced plasmid genomes, the resulting information remains rather limited. Using 503 sequenced plasmids organized in the ACLAME database, we discuss how, by structuring information on the genomes, their host and the proteins they code for, one can gain access to either global or more detailed analysis of the plasmid sequence information, as illustrated by a network representation of the relationships between plasmids.     

His-FemA融合蛋白表达载体的构建及其在原核细胞中的表达

目的 构建His标签的金黄色葡萄球菌甲氧西林耐药相关蛋白FemA的融合蛋白表达载体,并在大肠埃希菌中表达,为进一步研究femA基因的生物学功能和临床应用奠定基础.方法 根据GenBank中金黄色葡萄球菌femA基因序列,利用Primer Premier 5.0设计PCR引物,并在引物的5'加入BamHI及SalI酶切位点;以金黄色葡萄球菌基因组DNA为模版,PCR扩增出femA基因片段.将目的DNA片段及质粒pQE30分别进行双酶切、连接并转化大肠埃希菌DH5α;阳性克隆以PCR、双酶切及测序进行鉴定.将鉴定正确的pQE30-femA重组质粒转化大肠埃希菌JM109,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达His-femA融合蛋白;采用SDS-PAGE及Western blot分析对表达蛋白进行验证.结果 经PCR、双酶切签定及序列测定,证实重组质粒pQE30-femA构建成功;重组质粒pQE30-femA转化大肠埃希菌JM109经IPTG诱导后,SDS-PAGE和Western blot分析显示表达出53 kD目的蛋白;经Bandscan软件分析,目的蛋白质在4h的表达量占细胞总蛋白的27.5％.结论 成功构建了His-FemA原核表达载体(pQE30-femA),并在大肠埃希菌中高效表达.     

Proteomic analysis of global alteration of protein expression in squamous cell carcinoma of the esophagus

Squamous cell carcinoma of the esophagus (ESCC), a major subtype of esophageal carcinoma, is one of the aggressive cancers with worst prognosis in the world. The dismal outcome of ESCC is attributed to multiple reasons including its aggressive nature, largely unknown molecular mechanism of its progression, and the lack of biomarkers for early detection and effective prediction of its clinical behavior. To identify proteins with prognostic and/or predictive value, we applied a proteomics strategy to quantify proteins differentially expressed in ESCC using matched samples of carcinoma and adjacent normal epithelial cells. The analysis led to identification of 28 proteins aberrantly expressed in cancer cells with changes of at least three-fold in ESCC relative to normal squamous epithelial cells. These changes represent functional alterations of essential proteins for normal cellular physiology, accounting for many cellular changes involved in development of ESCC, including cell transformation, loss of differentiation, tumor growth, apoptosis, tumor invasion, and cell metabolism. The differentially expressed proteins shed new insights on the mechanism of tumorigenesis and provide candidate biomarkers for early detection of ESCC.     

GST-Ccd1融合蛋白的表达、纯化及多克隆抗体制备

目的：利用大肠杆菌DH5α表达GST—Ccd1融合蛋白,并用亲和层析分离纯化,进行动物免疫制备多克隆抗体。方法：利用本室构建好的pGEX-5X-1-Ccd1-N原核表达重组质粒,转化大肠杆菌DH5α,经IPTG诱导表达,在大肠杆菌表达系统中获得可溶性表达。经谷胱甘肽Sepharose 4B介质填充的层析柱分离纯化蛋白,制备抗原免疫动物,得到Ccd1的兔源多克隆抗体。结果：ELISA结果显示血清抗体效价可以达到1∶40 000。免疫组化分析表明自制的抗体能特异性与Ccd1蛋白相互作用,可以用于实验分析。结论：制备了效价高特异性良好的抗Ccd1多克隆抗体,经实验验证获得的抗体能够满足针对Ccd1的免疫印迹和免疫组化检测的实验要求,为今后深入研究Ccd1表达的组织分布、细胞内定位及其生物学功能提供了有用的实验工具。