Screening Of Expression Library Research Articles

Proteomics-based Expression Library Screening (PELS): A Novel Method for Rapidly Defining Microbial Immunoproteomes

Current methodologies for global identification of microbial proteins that elicit host humoral immune responses have several limitations and are not ideally suited for use in the postgenomic era. Here we describe a novel application of proteomics, proteomics-based expression library screening, to rapidly define microbial immunoproteomes. Proteomics-based expression library screening is broadly applicable to any cultivable, sequenced pathogen eliciting host antibody responses and hence is ideal for rapidly mining microbial proteomes for targets with diagnostic, prophylactic, and therapeutic potential. In this report, we demonstrate "proof-of-principle" by identifying 207 proteins of the Escherichia coli O157:H7 immunome in bovine reservoirs in only 3 weeks.

Characterization of a β‐N‐acetylhexosaminidase and a β‐N‐acetylglucosaminidase/β‐glucosidase from Cellulomonas fimi

The gram-positive soil bacterium Cellulomonas fimi is shown to produce at least two intracellular beta-N-acetylglucosaminidases, a family 20 beta-N-acetylhexosaminidase (Hex20), and a novel family 3-beta-N-acetylglucosaminidase/beta-glucosidase (Nag3), through screening of a genomic expression library, cloning of genes and analysis of their sequences. Nag3 exhibits broad substrate specificity for substituents at the C2 position of the glycone: kcat/Km values at 25 degrees C were 0.066 s(-1) x mM(-1) and 0.076 s(-1) x mM(-1) for 4'-nitrophenyl beta-N-acetyl-D-glucosaminide and 4'-nitrophenyl beta-D-glucoside, respectively. The first glycosidase with this broad specificity to be described, Nag3, suggests an interesting evolutionary link between beta-N-acetylglucosaminidases and beta-glucosidases of family 3. Reaction by a double-displacement mechanism was confirmed for Nag3 through the identification of a glycosyl-enzyme species trapped with the slow substrate 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside. Hex20 requires the acetamido group at C2 of the substrate, being unable to cleave beta-glucosides, since its mechanism involves an oxazolinium ion intermediate. However, it is broad in its specificity for the D-glucosyl/D-galactosyl configuration of the glycone: Km and kcat values were 53 microM and 482.3 s(-1) for 4'-nitrophenyl beta-N-acetyl-D-glucosaminide and 66 microM and 129.1 s(-1) for 4'-nitrophenyl beta-N-acetyl-D-galactosaminide.

Screening Expression Libraries Constructed in Plasmid Vectors

Screening Expression Libraries Constructed in Plasmid Vectors

Screening Expression Libraries Constructed in Bacteriophage λ Vectors

Screening Expression Libraries Constructed in Bacteriophage λ Vectors

CDNA expression library screening for identification of novel onconeural antigens

Paraneoplastic neurological disorders occur as a remote effect of cancer. Tumor cells expressing neuron-specific proteins elicit an autoimmune response, resulting in the production of various antibodies. The antibodies are usually associated with different syndromes, but the identity of many of the antigens is still unknown. Screening a cDNA expression library is a powerful technique that allows identification of previously uncharacterized antigens. By using patient sera containing antibodies, the antigens of interest can be isolated and further characterized.

LfhA, a novel factor H-binding protein of Leptospira interrogans.

The early phase of leptospiral infection is characterized by the presence of live organisms in the blood. Pathogenic Leptospira interrogans is resistant to the alternative pathway of complement mediated-killing, while nonpathogenic members of the genus are not. Consistent with that observation, only pathogenic leptospires bound factor H, a host fluid-phase regulator of the alternative complement pathway. Ligand affinity blot analyses revealed that pathogenic L. interrogans produces at least two factor H-binding proteins. Through screening of a lambda phage expression library, we identified one of these as the novel membrane protein LfhA. Ligand affinity assays and surface plasmon resonance analyses of recombinant LfhA revealed specific binding of both factor H and factor H-related protein 1. Serological examination of infected humans and horses demonstrated that LfhA is expressed by L. interrogans during mammalian infection. LfhA may therefore contribute to the resistance of pathogenic leptospires to complement-mediated killing during leptospiremic phases of the disease.

Serological immune response to cancer testis antigens in patients with pancreatic cancer

Serological screening approaches have allowed for the identification of a large number of potentially relevant tumor antigens in cancer patients. Within this group, cancer testis antigens represent promising targets for cancer immunotherapy, since they are widely expressed in a variety of human cancer entities. In pancreatic cancer, however, there are only few data available about the expression pattern and serological response to cancer testis antigens and other serological-defined tumor antigens. Therefore, we investigated the IgG antibody response against 11 cancer testis antigens (SCP-1, GAGE, LAGE-1a,-1b, CT-7, NY-ESO-1, SSX-1-5) recombinantly expressed on yeast surface (RAYS) in patients with pancreatic cancer (n = 96), chronic pancreatitis (n = 18) and healthy donors (n = 48). We found in 14% of all patients antibody responses to SCP-1, but not to other cancer testis antigens (GAGE, LAGE-1a,-1b, CT-7, NY-ESO-1, SSX-1-5). Antibody response correlated with the expression of SCP-1 in the primary tumor of the respective patient as shown by RT-PCR, immunohistochemistry and Western blot. In contrast, no serological response to cancer testis antigens was observed in healthy donors. The humoral immune response against SCP-1 was associated with the size of tumor, but not with other clinico-pathological parameters such as histology, stage, presence of lymph node metastases, grading, age, gender or gemcitabine treatment. In conclusion, antibody response to cancer testis antigen SCP-1 is found in a proportion of pancreatic carcinoma patients. These results indicate that identification of additional tumor antigens by serological screening of tumor cDNA expression libraries by RAYS is a promising goal in pancreatic cancer.

Identification of skeletal muscle autoantigens by expression library screening using sera from autoimmune rippling muscle disease (ARMD) patients

Novel forms of contractile regulation observed in skeletal muscle are evident in neuromuscular diseases like rippling muscle disease (RMD). Previous studies of an autoimmune form of RMD (ARMD) identified a very high molecular weight skeletal muscle protein antigen recognized by ARMD patient antisera. This study utilized ARMD and myasthenia gravis (MG) patient antisera, to screen a human skeletal muscle cDNA library that subsequently identified proteins that could play a role in ARMD. Based on nucleotide sequence analysis, three distinct ARMD antigens were identified: titin Isoform N2A, ATP synthase 6, and PPP1R3 (protein phosphatase 1 regulatory subunit 3). The region of titin identified by ARMD antisera is distinct from the main immunogenic region (MIR) recognized by classical MG antibodies. Sera from classical MG patient identifies an expressed sequence corresponding to the titin MIR. Although the mechanism of antibody penetration is not known, previous studies have shown that rippling muscle antibodies affect the contractile machinery of myofibers resulting in mechanical sensitivity. Titin's role as a modulator of muscle contractility makes it a potential target in understanding muscle mechanosensitive regulation.

Toward a protein map of the green algal eyespot: analysis of eyespot globule-associated proteins

S. Renninger, C.L. Dieckmann and G. Kreimer. 2006. Toward a protein map of the green algal eyespot: analysis of eyespot globule-associated proteins. Phycologia 45: 199–212. DOI: 10.2216/05-16.1Despite the well-established biophysical function of the green algal eyespot as a combined absorption/reflection screen for the retinal-based photoreceptors, only little is known about structural eyespot proteins and proteins involved in its assembly. A recently developed method for preparation of eyespot globules from Spermatozopsis similis (Chlorophyceae) has allowed now a detailed characterisation of this specific subset of eyespot proteins. Five antisera were raised against eyespot globule-associated proteins (GAPs). Two-dimensional gel electrophoresis and subsequent immunoblotting were combined to analyse the GAPs in detail. Approximately 180 spots were resolved in silver-stained two-dimensional gels, indicating the complexity of proteins needed for formation and maintenance of the highly ordered eyespot globule plate and its interactions with the chloroplast envelope and thylakoid. Topological analyses ide.gified 19 proteins possessing domains hidden in the globule matrix or otherwise not susceptible to thermolysin treatment. These GAPs might be involved in globule stabilisation. Antibody screening of a cDNA expression library resulted in the isolation of a full-length clone coding for a novel basic proline- and alanine-rich protein of 727 amino acids. The deduced amino acid sequence revealed the presence of a putative N-terminal signal peptide, followed by a hydrophobic region and four TPR m.gifs in the C terminal region. This protein, termed GAP1, might thus be involved in mediating protein–protein interactions in a protein scaffold around the hydrophobic globule core. This study paves the way for future molecular ide.gification of GAPs by systematic proteomic approaches.

A novel 78-kDa fatty acyl-CoA synthetase (ACS1) of Babesia bovis stimulates memory CD4 + T lymphocyte responses in B. bovis-immune cattle

Antigen-specific CD4 + T lymphocyte responses contribute to protective immunity against Babesia bovis, however the antigens that induce these responses remain largely unknown. A proteomic approach was used to identify novel B. bovis antigens recognized by memory CD4 + T cells from immune cattle. Fractions obtained from merozoites separated by continuous-flow electrophoresis (CFE) that contained proteins ranging from 20 to 83 kDa were previously shown to stimulate memory CD4 + lymphocyte responses in B. bovis-immune cattle. Expression library screening with rabbit antiserum raised against an immunostimulatory CFE fraction identified a clone encoding a predicted 78 kDa protein. BLAST analysis revealed sequence identity of this B. bovis protein with Plasmodium falciparum fatty acyl coenzyme A synthetase (ACS) family members (PfACS1–PfACS11), and the protein was designated B. bovis acyl-CoA synthetase 1 (ACS1). Southern blot analysis indicated that B. bovis ACS1 is encoded by a single gene, although BLAST analysis of the preliminary B. bovis genome sequence identified two additional family members, ACS2 and ACS3. Peripheral blood lymphocytes and CD4 + T cell lines from B. bovis-immune cattle proliferated significantly against recombinant ACS1 protein, consistent with its predicted involvement in protective immunity. However, immune sera from cattle recovered from B. bovis infection did not react with ACS1, indicating that epitopes may be conformationally dependent.

Proteomics‐based generation and characterization of monoclonal antibodies against human liver mitochondrial proteins

Monoclonal antibodies (mAbs) have the potential to be a very powerful tool in proteomics research to determine protein expression, quantification, localization and modification, as well as protein-protein interactions, especially when combined with microarray technology. Thus, a large amount of well-characterized and highly qualified antibodies are needed in proteomics. Purified antigen, which is not always available, has proven to be one of the rate-limiting steps in mAb large-scale generation. Here we describe our strategies to establish a murine hybridoma cell bank for human liver mitochondria using unknown native proteins as the immunogens. The antibody-recognized mitochondrial proteins were identified by MS following immunoprecipitation (IP), and by screening of human liver cDNA expression library. We found that the established antibodies reacted specifically with a number of important enzymes in mitochondria. The subcellular localization of these antigens in mitochondria was further confirmed by immunohistocytochemistry. A panel of antibodies was also tested for their ability to capture and deplete the targeting proteins and complexes from the total mitochondrial proteins. We believe these well-characterized antibodies would be useful in various applications for Human Liver Proteome Project (HLPP) when the scale of this hybridoma cell bank is enlarged significantly in the near future.

Profiling humoral autoimmune repertoire of dilated cardiomyopathy (DCM) patients and development of a disease‐associated protein chip

Dilated cardiomyopathy (DCM) is a myocardial disease characterized by progressive depression of myocardial contractile function and ventricular dilatation. Thirty percent of DCM patients belong to the inherited genetic form; the rest may be idiopathic, viral, autoimmune, or immune-mediated associated with a viral infection. Disturbances in humoral and cellular immunity have been described in cases of myocarditis and DCM. A number of autoantibodies against cardiac cell proteins have been identified in DCM. In this study, we have profiled the autoantibody repertoire of plasma from DCM patients against a human protein array consisting of 37,200 redundant, recombinant human proteins and performed qualitative and quantitative validation of these putative autoantigens on protein microarrays to identify novel putative DCM specific autoantigens. In addition to analyzing the whole IgG autoantibody repertoire, we have also analyzed the IgG3 antibody repertoire in the plasma samples to study the characteristics of IgG3 subclass antibodies. By combining screening of a protein expression library with protein microarray technology, we have detected 26 proteins identified by the IgG antibody repertoire and 6 proteins bound by the IgG3 subclass. Several of these autoantibodies found in plasma of DCM patients, such as the autoantibody against the Kv channel-interacting protein, are associated with heart failure.

T cell receptor-transgenic primary T cells as a tool for discovery of leukaemia-associated antigens

Identification of a broad array of leukaemia-associated antigens is a crucial step towards immunotherapy of haematological malignancies. However, it is frequently hampered by the decrease of proliferative potential and functional activity of T cell clones used for screening procedures. Transfer of the genes encoding the T cell receptor (TCR) alpha and beta chains of leukaemia-specific clones into primary T cells may help to circumvent this obstacle. In this study, transfer of two minor histocompatibility antigen (minor H antigen)-specific TCRs was performed and the feasibility of the use of TCR-transgenic T cells for identification of minor H antigens through cDNA library screening was investigated. We found that TCR-transgenic cells acquired the specificity of the original clones and matched their sensitivity. Moreover, the higher scale of cytokine-production by TCR-transgenic T cells permits the detection of either small amounts of antigen-positive cells or cells expressing low amounts of an antigen. When applied in equal numbers, TCR-transgenic T cells and the original T cell clones produced similar results in the screening of a cDNA library. However, the use of increased numbers of TCR-transgenic T cells allowed detection of minute amounts of antigen, barely discernible by the T cell clone. In conclusion, TCR-transfer generates a large amount of functional antigen-specific cells suitable for screening of cDNA expression libraries for identification of cognate antigens.

Human ribosomal protein L18a interacts with hepatitis C virus internal ribosome entry site

Translation initiation of hepatitis C virus RNA occurs via ribosome binding to an 'internal ribosome entry site (IRES)' located in the 5'untranslated region of the viral RNA. The principle interaction between the 40S ribosomal subunit and the HCV IRES has been shown to be largely factor independent, which is followed by the joining of the 60S ribosomal subunit to form functional 80S complex. However several additional cellular proteins have been reported to bind to HCV IRES and enhance the initiation of translation. In order to identify novel factors involved in the ribosome assembly during internal initiation of HCV RNA, northwestern screening of a HeLa cDNA expression library was performed, using HCV IRES RNA as probe. We demonstrate here, that human ribosomal protein L18a, a constituent of 60S subunit, interacts with HCV IRES RNA. This interaction was further confirmed by using a recombinant protein similar to L18a (sL18a), cloned from human blood. Interestingly, addition of increasing concentration of the purified recombinant sL18a protein, showed moderate stimulation of HCV IRES activity in the in vitro translation assay. These observations suggest that the human L18a might influence the HCV IRES mediated translation.

Anti-tumor activity of dendritic cells transfected with mRNA for receptor for hyaluronan-mediated motility is mediated by CD4+ T cells.

Receptor for hyaluronan-mediated motility (RHAMM) is overexpressed in various tumors with high frequency, and was recently identified as an immunogenic antigen by serologic screening of cDNA expression libraries. In this study, we explored whether RHAMM is a potential target for dendritic cell (DC) immunotherapy. We constructed a plasmid for transduction of in vitro-transcribed mRNAs into DCs to efficiently transport the intracellular protein RHAMM into MHC class II compartments by adding a late endosomal/lysosomal sorting signal to the RHAMM gene. Immunization of mice with modified RHAMM mRNA-transfected DCs (DC/RHAMM) induced killing activity against RHAMM-positive tumor cells in splenocytes. To examine whether CD4(+) and/or CD8(+) T cells were required for this antitumor immunity, an anti-CD4 or anti-CD8 antibody was administered to mice after immunization with DC/RHAMM. Depletion of CD4(+) T cells significantly diminished the induction of tumor cell-killing activity in splenocytes, whereas CD8(+) T cell depletion had no effect. We then investigated the therapeutic effect of DC/RHAMM in a 3-day tumor model of EL4. DC/RHAMM was administered to mice on days 3, 7 and 10 after EL4 tumor inoculation. The treatment markedly inhibited tumor growth compared to control DCs. Moreover, antibody-mediated depletion of CD4(+) T cells completely abrogated the therapeutic effect of DC/RHAMM, whereas depletion of CD8(+) T cells had no effect. The results of this preclinical study indicate that DCs transfected with a modified RHAMM mRNA targeted to MHC class II compartments can induce CD4(+) T cell-mediated antitumor activity in vivo.

Cloning and characterization of a novel immunogenic protein 3 (NIP3) from Brugia malayi by immuno screening of a phage-display cDNA expression library

Iterative screening of a phage display cDNA expression library of the third-stage larvae (L3) of Brugia malayi with sera from putatively immune individuals (endemic normal, EN) identified a novel clone with insert showing significant homology to Onchocerca volvulus novel immunogenic protein-3 (Ov-NIP3) gene and Caenorhabditis elegans NIP3-like protein and hence the gene was designated Brugia malayi NIP3-like protein (BmNIP3). EST database analysis showed that ESTs from several gastrointestinal nematodes such as Ancylostoma caninum, Teladorsagia circumcincta, Haemonchus contortus and Strongyloides stercoralis has BmNIP3 homologues, but the gene has not been described from these parasites. Sequence analyses showed that BmNIP3 has three potential mucin-type O-glycosylation sites and several serine/threonine phosphorylation sites. As expected, BmNIP3 protein isolated from the parasite was serine/threonine phosphorylated. Further analyses showed that BmNIP3 is differentially transcribed, with highest level of expression present in the larval (L3 and L4) stages. Mice immunized with rBmNIP3 developed strong antibody responses predominantly of the IgG1 and IgG2a subtype. A similar analyses of the sera samples from EN individuals showed that they also carry high levels of IgG1 and IgG2 antibodies against BmNIP3, whereas, chronically infected patients carry largely IgG3 antibodies and MF individuals carry high levels of IgG1 antibodies against BmNIP3. This study thus describes a novel protein from B. malayi that appears to be highly immunogenic in both humans and mice.

Characterization of a novel vaccine candidate and serine proteinase inhibitor from Schistosoma japonicum (Sj serpin)

Serine proteinase inhibitors (serpins) represent an important superfamily of endogenous inhibitors that regulate proteolytic events active in a variety of physiological functions. Immunological screening of a Schistosoma japonicum adult worm cDNA expression library with sera of Microtus fortis, a naturally resistant vertebrate host, has identified one clone that encoded for a sequence homologous to those of the serpin superfamily. The full-length sequence encoding S. japonicum serpin (Sj serpin) was amplified from adult worm cDNA by using 5′-RACE-PCR and subsequently cloned into the prokaryotic expression vector pET28c. The full-length Sj serpin fusion-protein with his-tag was expressed in E. coli, purified by affinity chromatography and used to immunize New Zealand white rabbits. Sj serpin is located on the tegument in S. japonicum adult worms. C57BL/6 mice immunized with Sj serpin induced the production of high levels of specific IgE and IgG1 subclass antibodies as well as a marked IL-4 response. Lymphocyte surface marker analysis revealed proliferation of CD19 expressing B cells, indicating a predominant Th2-type response to Sj serpin. Immunized mice developed moderate protection against infection of S. japonicum as demonstrated by a 36 and 39% reduction in the recovery of adult worms and eggs, respectively. These data suggested a role for Sj serpin as a vaccine candidate or as a novel target for anti-schistosome drugs.

NADP-malic Enzyme and Hsp70: Co-purification of Both Proteins and Modification of NADP-malic Enzyme Properties by Association with Hsp70

Different preparations of antibodies against 62 kDa NADP-malic enzyme (NADP-ME) from purified maize leaves cross-react with a 72 kDa protein from diverse tissues in many species. A 72 kDa protein, suggested to be a non-photosynthetic NADP-ME, has been purified from several plant species. However, to date, a cDNA coding for this putative 72 kDa NADP-ME has not been isolated. The screening of maize and tobacco leaf expression libraries using antibodies against purified 62 kDa NADP-ME allowed the identification of a heat shock protein (Hsp70). In addition, tandem mass spectrometry (MS/MS) studies indicate that along with NADP-ME, a 72 kDa protein, identified as an Hsp70 and reacting with the antibodies, is also purified from maize roots. On the other hand, the screening of a maize root cDNA library revealed the existence of a cDNA that encodes a mature 66 kDa NADP-ME. These results suggest that the 72 kDa protein is not actually an NADP-ME but in fact an Hsp70, at least in maize and tobacco. Probably, NADP-ME-Hsp70 association, taking place at least when preparing crude extracts, can lead to a co-purification of the proteins and can thus explain the cross-reaction of the antibodies. In the present work, we analyse and discuss a probable interaction of NADP-ME with Hsp70.

A Novel Genomic Approach Identifies Bacterial DNA-Dependent RNA Polymerase as the Target of an Antibacterial Oligodeoxynucleotide, RBL1

Rapid emergence of antibiotic-resistant bacterial pathogens limits the applicability of existing drugs, which has created an urgent need for novel antibiotics preferably with entirely new mechanisms of action. Oligodeoxynucleotides (ODNs) and their modified forms have been shown to inhibit bacterial gene expression, representing a potential for developing highly specific and efficacious antibacterial agents. In this study, a tetracycline (Tet)-inducible, randomized single-stranded DNA (ssDNA) expression library was constructed and screened for conditional growth-defective or lethal phenotypes in an Escherichia coli system. From approximately 5000 transformants screened, 12 bacterial colonies were identified with either growth-defective or lethal phenotypes. One clone, CY01, with a lethal phenotype was selected and sequenced, and the ODN sequence that it generates was designated as RBL-1. Because RBL-1 shows no significant homologies to any bacterial gene sequence, a potential RBL-1 targeting protein was isolated by affinity purification. Using mass spectrometry analysis, this protein was identified as bacterial DNA-dependent RNA polymerase (RNAP). RBL-1 was further shown to effectively inhibit RNA polymerase activity in vitro. The usage of this randomized ssDNA expression library screening technology to selectively modulate production and/or function of proteins may provide a powerful strategy in both identifying novel gene targets for antibiotic discovery and developing novel antibacterial agents.

Identification of a 40S Ribosomal Protein S4–Derived H-Y Epitope Able to Elicit a Lymphoblast-Specific Cytotoxic T Lymphocyte Response

The superior graft-versus-leukemia (GVL) effect of the female-to-male stem cell transplantation is partially independent from the concomitant graft-versus-host reactivity. However, the antigenic basis of this selective GVL response remains enigmatic, because no H-Y antigens with hematopoietic-restricted expression were identified. In this study, we report a novel H-Y epitope that is preferentially recognized on activated proliferating lymphocytes. We generated a CTL clone YKIII.8 that showed reactivity toward male B*5201+ CD40-activated B cells, EBV-lymphoblastoid cell lines, and phytohemagglutinin-activated T-cell blasts but little or no reactivity toward fibroblasts, CD14+ cells, or unstimulated B and T cells. The antigen recognized by YKIII.8 was identified by screening of a cDNA expression library, and its pattern of expression was investigated. cDNA of the male isoform of 40S ribosomal protein S4 was found to encode the antigenic peptide TIRYPDPVI, which was recognized by YKIII.8. Western blot analysis showed that rapidly proliferating cells overexpress the RPS4 protein in comparison with nonrecognized cell subsets. Retroviral transfer of YKIII.8 T-cell receptor resulted in preservation of the lymphoblast-specific reactivity pattern. Our findings suggest that CTL specific to certain epitopes of ubiquitously expressed H-Y antigens may specifically target lymphoblasts, contributing to the selective GVL effect of female-to-male stem cell transplantation.