Identification of Amino Acids Essential for Antibody Binding by mRNA-Display Using a Random Peptide Library: An Anti-Human Tumor Protein p53 Antibody as a Model

Using a synthetic DNA library coding for random 10-amino acid peptides (R10aPL), mRNA-display was applied to the isolation of interactive peptides using a monoclonal antibody against human TP53 (hTP53) as a model. Display molecules consisting of peptides and the nucleotide sequences encoding them were synthesized in vitro and subjected to four to five cycles of affinity selection. Thirty-four clones each isolated in the 4th or 5th round were sequenced. A core sequence, (X)-S-D-L-(Z)-K-L essential for binding was found, in which (X) and (Z), though undefined, were mostly F or Y and W, respectively. Although no peptides that fully matched with hTP53 were found in the clones isolated, the core sequence was found in hTP53. A 10-amino acid peptide containing the core sequence was chemically synthesized to verify its binding with SPR. Its Kd value for the antibody was 6 nM. The amino acids in epitopes essential for binding could be identified by mRNA-display with R10aPL.     

The metal tolerance profile of <Emphasis Type="Italic">Thlaspi goesingense</Emphasis> is mimicked in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>heterologously expressing serine acetyl-transferase

Background  

The Ni hyperaccumulator Thlaspi goesingense is tolerant to Ni ≅ Zn, ≅ Co and slightly resistant to > Cd. We previously observed that elevated glutathione, driven by constitutive activation of serine acetyltransferase (SAT), plays a role in the Ni tolerance of T. goesingense.     

Molecular cloning,characterization and expression analysis of adenylosuccinate lyase gene in grass carp (<Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>)

Adenylosuccinate lyase (ADSL) is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling. In the present study, we have constructed a grass carp (Ctenopharyngodon idella) intestinal cDNA library that has over 2.3 × 10⁵ primary clones. An expressed sequence tag (EST) of grass carp adenylosuccinate lyase (gcADSL) gene was screened from this library. Both 5′-RACE and 3′-RACE were carried out in order to obtain the complete cDNA sequence, which contains a 1,446 bp open reading frame encoding 482 amino acids about 54.552 kDa. The deduced amino acid sequence shares high homology with its vertebrate counterparts, which shares 94% similarity with zebrafish, 81% with African clawed frog as well as chicken, 77% with human and 76% with mouse. This gcADSL genomic sequence, consisted of 13 exons and 12 introns, is 8,557 bp in size. Real-time quantitative PCR analysis revealed that the highest expression level of gcADSL was detected in muscle and the lowest in gill. In western blotting analysis, His₆-tagged gcADSL protein expressed in Escherichia coli could be recognized not only by an anti-His₆-tag monoclonal antibody but also by an anti-human ADSL polyclonal antibody, indicating immunological crossreactivity occurs between grass carp and human ADSL protein. 1,082 bp 5′-flanking region sequence was cloned and analyzed.     

Antibody responses to galectin-8, TARP and TRAP1 in prostate cancer patients treated with a GM-CSF-secreting cellular immunotherapy

A critical factor in clinical development of cancer immunotherapies is the identification of tumor-associated antigens that may be related to immunotherapy potency. In this study, protein microarrays containing >8,000 human proteins were screened with serum from prostate cancer patients (N = 13) before and after treatment with a granulocyte–macrophage colony-stimulating factor (GM-CSF)-secreting whole cell immunotherapy. Thirty-three proteins were identified that displayed significantly elevated (P ≤ 0.05) signals in post-treatment samples, including three proteins that have previously been associated with prostate carcinogenesis, galectin-8, T-cell alternative reading frame protein (TARP) and TNF-receptor-associated protein 1 (TRAP1). Expanded analysis of antibody induction in metastatic, castration-resistant prostate cancer (mCRPC) patients (N = 92) from two phase 1/2 trials of prostate cancer immunotherapy, G-9803 and G-0010, indicated a significant (P = 0.03) association of TARP antibody induction and median survival time (MST). Antibody induction to TARP was also significantly correlated (P = 0.036) with an increase in prostate-specific antigen doubling time (PSADT) in patients with a biochemical (PSA) recurrence following prostatectomy or radiation therapy (N = 19) from in a previous phase 1/2 trial of prostate cancer immunotherapy, G-9802. RNA and protein encoding TARP and TRAP1 was up-regulated in prostate cancer tissue compared to matched normal controls. These preliminary findings suggest that antibody induction to TARP may represent a possible biomarker for treatment response to GM-CSF secreting cellular immunotherapy in prostate cancer patients and demonstrates the utility of using protein microarrays for the high-throughput screening of patient-derived antibody responses.     

Identification of a Novel Vanadium-binding Protein by EST Analysis on the Most Vanadium-rich Ascidian, Ascidia gemmata

Ascidians are known to accumulate extremely high levels of vanadium in their blood cells (up to 350 mM). The branchial sac and the intestine are thought to be the first tissues to contact the outer environment and absorb vanadium ions. The concentration of vanadium in the branchial sac and the intestine of the most vanadium-rich ascidian Ascidia gemmata were determined to be 32.4 and 11.9 mM, respectively. Using an expressed sequence tag (EST) analysis of a cDNA library from the intestine of A. gemmata, we determined 960 ESTs and found 55 clones of metal-related gene orthologs, 6 redox-related orthologs, and 18 membrane transporter orthologs. Among them, two genes, which exhibited significant similarity to the vanadium-binding proteins of other vanadium-rich ascidian species, were designated AgVanabin1 and AgVanabin2. Immobilized metal ion affinity chromatography revealed that recombinant AgVanabin1 bound to metal ions with an increasing affinity for Cu(II) > Zn(II) > Co(II) and AgVanabin2 bound to metal ions with an increasing affinity for Cu(II) > Fe(III) > V(IV). To examine the use of AgVanabins for a metal absorption system, we constructed Escherichia coli strains that expressed AgVanabin1 or AgVanabin2 fused to maltose-binding protein and secreted into the periplasmic space. We found that the strain expressing AgVanabin2 accumulated about 13.5 times more Cu(II) ions than the control TB1 strain. Significant accumulation of vanadium was also observed in the AgVanabin2-expressing strain as seen by a 1.5-fold increase.     

mRNA-display-based selections for proteins with desired functions: a protease-substrate case study

mRNA-display is an amplification-based, iterative rounds of in vitro protein selection technique that circumvents a number of difficulties associated with yeast two-hybrid and phage display. Because of the covalent linkage between the genotype and the phenotype, mRNA-display provides a powerful means for reading and amplifying a peptide or protein sequence after it has been selected from a library with a diversity in the range of 10(12)-10(13). In this paper, we briefly review the recent progress in using mRNA-display to identify affinity reagents, binding partners, and enzyme substrates from synthetic peptide or natural proteome libraries. To facilitate the use of mRNA-display in research laboratories without previous experience, we provide a detailed analysis of the critical steps of an mRNA-display-based selection in a case study for the identification of the natural substrates of caspases, including the generation of an mRNA-displayed proteome library, removal of abundant sequences, and selection of proteins with desired functions. The advantages and technical limitations of mRNA-display as a general peptide or protein selection tool are also addressed.     

An altered camelid-like single domain anti-idiotypic antibody fragment of HM-1 killer toxin: acts as an effective antifungal agent

Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species. The alternative small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data showed that the binding affinity for this single-domain antibody was 272-fold higher (K _d = 1.07 × 10⁻¹⁰ M) and antifungal activity was three- to fivefold more efficient (IC₅₀ = 0.46 × 10⁻⁶ to 1.17 × 10⁻⁶ M) than that for the conventional antibody (K _d = 2.91 × 10⁻⁸ M and IC₅₀ = 2.14 × 10⁻⁶ to 3.78 × 10⁻⁶ M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain synthetic antibodies will find their way into a number of biotechnological or medical applications.     

Serum p53 protein and anti-p53 antibodies are associated with increased cancer risk: a case–control study of 569 patients and 879 healthy controls

The aim of this study to determine whether serum p53 protein and antibodies are associated with malignant tumors. A case–control study was conduct in 569 patients with various types of malignant tumors and 879 healthy controls. Serum p53 protein and antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA).The rate of positive p53 protein in patients with various malignant tumors was 4.22% compared with 0.34% in healthy controls (P < 0.001). The rate of anti-p53 antibodies in patients with various malignant tumors was 14.59% compared with 1.02% in healthy controls (P < 0.001). The adjusted odd ratio (OR) for p53 protein was 17.55 (95% CI = 4.98–61.94). The adjusted odd ratio for anti-p53 antibodies was 14.27 (95% CI = 6.75–30.16). The study strongly suggested that serum p53 protein and antibody are associated with increased cancer risk and can be used as early serological markers in the diagnosis of malignancies tumors.     

Molecular cloning and expression of a Schistosoma japonicum tegumental membrane-associated antigen from Japanese strain

Diagnosis and vaccine development form the major focus in creating strategies for the control of schistosomiasis. In this study, we established an IgG1 mouse monoclonal antibody (MoAb), SJA111, which strongly reacted with 23–25-kDa Schistosoma japonicum tegumental-associated membrane proteins, but not with eight other parasitic antigens. A λgt 11 cDNA library from the Japanese strain of the Schistosoma japonicum adult worm was screened with SJA111 as a probe. A single positive clone was isolated and the nucleotide sequence of the isolated cDNA was determined. The cDNA clone consisted of 844 bp, and the coding region contained 576 bp which was translated to a 22.6-kDa protein. This region showed 99.0% and 99.3% significant homology with those of the Chinese and Philippine strains of Schistosoma japonicum, respectively. The deduced amino acid sequence of the protein was identical to that of the Philippine strain and only one residue differed from that of the Chinese strain. The recombinant form of the tegumental protein was expressed in Escherichia coli and purified by a combination of ion exchange and affinity chromatography, and the purified protein was found to react with the sera of patients infected with Schistosoma japonicum. This result suggests that this antigen may be useful in the immunodiagnosis of schistosomiasis as well as in the development of an effective vaccine.     

Characterization of a cDNA clone encoding a new species of the nonspecific cross-reacting antigen (NCA), a member of the CEA gene family

To clarify the molecular structures of the nonspecific cross-reacting antigens (NCAs) produced by human granulocytes, we cloned cDNAs from libraries of normal white blood cells. A clone, NCA-W272, was found to code a protein similar to NCA of tumor cells. The protein consisted of a signal peptide (34 aa), domain-N (108 aa), -A1 (92 aa), -B1 (86 aa) and -M (29 aa). Similarity of the amino acid sequence of each domain to that of the tumor NCA was 72, 92, 76 and 79%, respectively. COS-1 cells transfected with an expression vector carrying the cDNA synthesized a 70 kDa glycoprotein, which was reactive with anti-NCA antibody and released from cell surface by phosphatidylinositol-specific phospholipase C. Thus the clone NCA-W272 was indicated to encode a new species of NCA distinct from the tumor NCA.     

Three different NCA species, CGM6/CD67, NCA-95, and NCA-90, are comprised in the major 90 to 100-kDa band of granulocyte NCA detectable upon SDS-polyacrylamide gel electrophoresis.

Human granulocytes express several species of nonspecific cross-reacting antigens (NCA), glycoproteins belonging to the carcinoembryonic antigen (CEA) family. Our previous studies have shown that at least two different NCA of 95 and 90 kDa are contained in the major NCA band of 90 to 100 kDa detectable upon gel electrophoresis of immunoprecipitates obtained from the cell surfaces of granulocytes with polyclonal anti-NCA. In the present study, the 90 to 100-kDa NCA band was found to include one more species of 100 kDa. This component was reactive with an anti-CD67 antibody as well as polyclonal anti-NCA and released from the cell surface with phosphatidylinositol-specific phospholipase C, indicating that the 100-kDa NCA species is CD67. Both antibodies revealed high binding activities with a recombinant protein of CGM6, which has been identified in a leukocyte cDNA library as an NCA gene and found to encode a glycosyl-phosphatidylinositol-anchored heterotypic cell adhesion molecule. Furthermore, the apparent molecular mass of the deglycosylated CD67 (38 kDa) corresponded with that of the CGM6 protein. These results suggest that CD67 is equivalent to the NCA species CGM6.     

Generation of Arabidopsis Mutants by Heterologous Expression of a Full-Length cDNA Library from Tomato Fruits

Heterologous expression of cDNA library in Arabidopsis and other plants has been used for gene identifications. To identify functions of tomato genes, we expressed a tomato full-length cDNA library in Arabidopsis thaliana and generated over 7,000 mutants. We constructed a tomato cDNA library with a plant transformation-ready binary vector that contained a higher percentage of full-length cDNAs since synthesized double-stranded cDNA was size-selected using gel electrophoresis, with cDNA sizes of 2–5 kb being gel-purified for ligation onto the binary vector. Sequencing of 81 cDNA clones indicates that 75% (61) are full-length genes, which is similar to sequencing of inserted cDNA in Arabidopsis. The library was used to transform Arabidopsis plants. Among the 7,000 mutants, one was found to be a dwarf due to the expression of an ATP synthase, and another vegetative mutant did not produce flowers even after 7 months. The technique was validated by reintroducing the tomato ribosomal protein L9 gene and can be used in any other plant species as a gene discovery tool.     

High-quality RNA preparation for cDNA library construction of the Antarctic sea–ice alga <Emphasis Type="Italic">Chlamydomonas</Emphasis> sp. ICE-L

To study the molecular mechanism of the Antarctic sea–ice alga in adaptation to polar sea–ice environments, the RNA was prepared for cDNA library construction of Chlamydomonas sp. ICE-L. Three different methods were tested to prepare total RNA from this psychrophilic, unicellular green alga rich in protein and polysaccharide. Lauryl sodium sulfate- based method allowed a most effective extraction of high-quality total RNA compared to the other methods. Total RNA extracted with this protocol was used for cDNA library construction. The recombination rate of constructed cDNA library was 98.60%, the primary titer was 7.15 × 10⁶ pfu, and an average sequence length was 1.2 kb. These results show that with a high-quality RNA preparation, a cDNA library can be constructed successfully for Chlamydomonas sp. ICE-L.     

Characterization of Various Recombinant Antigens from <Emphasis Type="Italic">Echinococcus multilocularis</Emphasis> for Use in the Immunodiagnosis

Using four clones isolated from Echinococcus multilocularis cDNA library with alveolar echinococcosis (AE) patient sera, various antigens were expressed as ThioHis tag-fused protein. Recombinant EmII/3 antigen was produced as the five fragments divided into the N-terminal (#5 and #5s), the central (#6 and #6s) and the C-terminal domain (#7). Immunoblot analysis revealed that the #7 showed significant reactivity whereas those of #5 and #5s were relatively low. The #6 and #6s also showed lower reactivity than that of #7, although the two minor bands of #6 reacted with every serum. These results suggested that an immunodominant region of EmII/3 locate within the C-terminal one third. The #8s recombinant antigen, Ser²³–Glu¹⁷⁶ of actin filament fragmenting protein (AFFP), apparently reacted with the AE patient sera, while the #1 antigen synthesized as a full-length antigen B1 did not show such high reactivity. Thus, #7 and #8s antigens showed significant potential for use in immunodetection of AE. In addition, the specific antibodies against #7 and #8s reacted with specific antigens in crude extract of E. multilocularis cyst, indicating that these antigens retained antigenicity common to native EmII/3 and AFFP, respectively.     

Determination of the cDNA sequence for the human mitochondrial 75-kDa Fe-S protein of NADH-coenzyme Q reductase

A human-hepatoma cDNA lambda gt11 expression library was probed with an antibody to holoenzyme complex I (NADH-CoQ reductase) of the respiratory chain. One of the 30 antibody positive clones was purified to homogeneity, amplified by the polymerase chain reaction (PCR), subcloned and sequenced. It proved to be highly similar to the cDNA sequence for the bovine 75-kDa Fe--S protein. Using the sequence obtained from this library, both sense and antisense oligonucleotides were constructed and used to probe a human kidney cDNA library using PCR amplification with oligonucleotides that flank the polylinker region of the lambda phage. Two further cDNA clones were obtained which overlapped and covered the entire cDNA sequence of 2526 bp. The encoded protein of 727 amino acids has 21 amino acids that differ from the bovine-protein sequence. Northern blot analysis of mRNA from fibroblasts of complex-I deficient patients revealed no abnormalities. We show that this Fe--S protein has significant similarity with (a) the gamma chain of the hydrogen hydrogenase of Alcaligenes eutrophus and (b) the A chain of the formate dehydrogenase of Methanobacterium formicum.     

Trypanosoma cruzi: cellular and antibody response against the parasite in mice immunized with a 19-amino acid synthetic peptide

Several monoclonal antibodies were prepared against the flagellar fraction of Trypanosoma cruzi epimastigotes (Tulahuén strain, stock Tul 2). One of them, FCH-F8-4, has previously shown biologic activity against the parasite (complement-mediated lysis and neutralization of the trypomastigote infectivity). Immunopurified antigens using this monoclonal antibody elicited a protective immune response in mice. Two recombinant cDNA clones were detected with this anti-flagellar fraction monoclonal antibody on a lambda gt11 expression library prepared from T. cruzi epimastigote mRNA. The insert of one of these cDNA clones, lambda(FCH-F8-4)1 (150 bp) coded for a 19-amino acid peptide (PAFLGCSSRFSGSFSGVEP). This insert hybridized with a 5.0-kb mRNA from epimastigotes. The beta-galactosidase fusion protein was produced in lysogenic bacteria. The monoclonal antibody recognized the epitope present in the fusion protein after western blotting of the crude lysate. A synthetic peptide (SP4) containing the complete sequence of lambda(FCH-F8-4)1 was constructed on solid phase. This peptide was able to inhibit the ELISA reactivity (in a range from 13 to 52%) of flagellar fraction immunized mouse sera and when administered (coupled to KLH or alone) to BALB/c mice with Bordetella pertussis as adjuvant, it induced a humoral and cellular immune response which was detected by ELISA, immunofluorescence, blotting, and DTH reactions against T. cruzi antigens. The immune response obtained indicates that this synthetic peptide resembles the parasite antigen conformation and could be useful for diagnosis purposes or be able to elicit immunoprotection against T. cruzi infection.