Human recombinant neutralizing antibodies against hantaan virus G2 protein.

Old world hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS), still present a public health problem in Asia and Eastern Europe. The majority of cases has been recorded in China. The aim of our study was to generate human recombinant neutralizing antibodies to a hantavirus by phage display technology.

Generation of an HFRS patient-derived neutralizing recombinant antibody to Hantaan virus G1 protein and definition of the neutralizing domain.

Hantaan virus (HTNV) in the Hantavirus genus, family Bunyaviridae, is the major cause of severe hemorrhagic fever with renal syndrome (HFRS). We prepared a combinatorial phage display library of human Fabs to HTNV from RNA extracted from the blood lymphocytes of a convalescent HFRS patient. We selected two G1 glycoprotein-specific clones and one nucleocapsid protein (N)-specific clone from the Fab library for further studies.

Mutation in the ARH gene and a chromosome 13q locus influence cholesterol levels in a new form of digenic-recessive familial hypercholesterolemia.

We studied a Syrian family with 3 children who had low-density lipoprotein cholesterol (LDL) concentrations of 13.3, 12.2, and 8.

Baculovirus expression cassette vectors for rapid production of complete human IgG from phage display selected antibody fragments.

For the expression of human intact IgG antibodies, we have constructed a set of baculovirus expression vectors designed to facilitate rapid insertion of heavy and light chain genes of Fab or scFv antibodies derived from phage display antibody libraries. By linking them to human constant or Fc regions, expression of complete human immunoglobulin molecules was achieved in insect cells by infection with recombinant baculovirus. The IgG expression cassette vectors are based on the backbone vector which contains two back to back polyhedron and p10 promoters.

Fine mapping of the antigen-antibody interaction of scFv215, a recombinant antibody inhibiting RNA polymerase II from Drosophila melanogaster.

A bacterially expressed single chain antibody (scFv215) directed against the largest subunit of drosophila RNA polymerase II was analysed. Structure and function of the antigen binding site in scFv215 were probed by chain shuffling and by site-specific mutagenesis. The entire variable region of either the heavy or light chain was replaced by an unrelated heavy or light chain.

A minimal binding domain of the low density lipoprotein receptor family.

As more relatives of the low density lipoprotein receptor (LDLR) are discovered, defining their minimal binding domain(s) becomes a challenge. Here we have chosen the multifunctional chicken oocyte receptor for yolk deposition (termed LR8), and the pan-receptor ligand, receptor associated protein (RAP), as model systems to characterize a minireceptor using the phage display approach. Displayed fragments derived from the entire 819 residue LR8 molecule, followed by selection via panning on RAP, led to the definition of an 80 residue stretch LR8 minireceptor.

Epitope mapping by phage display: random versus gene-fragment libraries.

We present a comparative study on epitope mapping of four monoclonal antibodies directed against four different antigens using alternative phage display techniques and peptide scanning: mAb215 reacts with the largest subunit of RNA polymerase II, mAbBp53-11 with the tumor suppressor protein p53, mAbGDO5 with the Hantaan virus glycoprotein G2 and mAbL13F3 with the Hantaan virus nucleocapsid protein. Epitopes were determined (i) by gene-fragment phage display libraries, constructed by DNaseI digested random gene fragments cloned into the 5' terminus of the pIII-gene of fd phage and (ii) by random peptide phage libraries displaying 6mer and 15mer peptides at the N-terminus of the pIII protein. Using the gene-fragment phage display libraries a single round of affinity selection resulted in the determination of the corresponding epitopes for all monoclonal antibodies tested.

Identification of the gene coding for the largest subunit of RNA polymerase I (A) of Drosophila melanogaster.

The gene coding for the largest subunit (RPA1) of RNA polymerase I (A) of Drosophila melanogaster (DmRPA1) was cloned and sequenced. The gene is interrupted by seven small introns and the cDNA reveals an open reading frame of 4932 nucleotides. The deduced polypeptide consists of 1644 amino acids with a calculated molecular weight of 185 kDa.

A major antigenic domain of hantaviruses is located on the aminoproximal site of the viral nucleocapsid protein.

Hantavirus nucleocapsid protein has recently been shown to be an immunodominant antigen in hemorrhagic with renal syndrome (HFRS) inducing an early and long-lasting immune response. Recombinant proteins representing various regions of the nucleocapsid proteins as well as segments of the G1 and the G2 glycoproteins of hantavirus strains CG18-20 (Puumala serotype) and Hantaan 76-118 have been expressed in E. coli.

Mapping of epitopes recognized by PM/Scl autoantibodies with gene-fragment phage display libraries.

Sera from patients suffering from the polymyositis/scleroderma overlap syndrome (PM/Scl) recognize two antigenically non-related proteins with apparent molecular masses of 100 kDa and 75 kDa respectively. The two proteins are part of a particle termed PM/Scl localized in the granular component of the nucleolus. The predominant immunoreactivity of the PM/Scl sera was shown to be directed against the 100 kDa protein.

Localization of yeast RNA polymerase I core subunits by immunoelectron microscopy.

Immunoelectron microscopy was used to determine the spatial organization of the yeast RNA polymerase I core subunits on a three-dimensional model of the enzyme. Images of antibody-labeled enzymes were compared with the native enzyme to determine the localization of the antibody binding site on the surface of the model. Monoclonal antibodies were used as probes to identify the two largest subunits homologous to the bacterial beta and beta' subunits.

Mapping of linear epitopes recognized by monoclonal antibodies with gene-fragment phage display libraries.

Epitope mapping with mono- or polyclonal antibodies has so far been done either by dissecting the antigens into overlapping polypeptides in the form of recombinantly expressed fusion proteins, or by synthesizing overlapping short peptides, or by a combination of both methods. Here, we report an alternative method which involves the generation of random gene fragments of approximately 50-200 bp in length and cloning these into the 5' terminus of the protein III gene of fd phages. Selection for phages that bind a given monoclonal antibody and sequencing the DNA inserts of immunopositive phages yields derived amino acid sequences containing the desired epitope.

Characterization of the epitope recognized by a monoclonal antibody directed against the largest subunit of Drosophila RNA polymerase II.

The epitope recognized by monoclonal antibody MAb215 generated previously against Drosophila melanogaster RNA polymerase II was mapped to amino acid residues 806-820 of the largest, 215 kDa, subunit located in a region conserved within the largest subunits of pro- and eukaryotic RNA polymerases. The affinities of MAb215 and of a recombinant single-chain Fv fragment (scFv215) were determined for binding to the enzyme as well as the fusion protein and synthetic peptides used for epitope mapping. In addition, amino acid residues of the epitope important for binding to MAb215 were identified using peptides carrying single amino acid substitutions.

Coding strategy of the S and M genomic segments of a hantavirus representing a new subtype of the Puumala serotype.

The hantavirus strain Vranica was previously reported to have been isolated from a bank vole in Bosnia-Hercegovina and associated with the occurrence of hemorrhagic fever with renal syndrome (HRFS) in humans. The complete cDNA nucleotide sequences of the small (S) and medium (M) genomic RNA segments of this virus were determined. Major open reading frames were found in the S and M segment between nucleotide positions 43 and 1341 coding for a polypeptide of 433 amino acid residues and between nucleotide positions 41 and 3,484 coding for 1,148 amino acid residues, respectively.

Nucleic acid-binding regions of the second-largest subunit of Drosophila RNA polymerase II identified by southwestern blotting.

Analysing overlapping bacterially expressed fragments of the second-largest subunit of Drosophila melanogaster RNA polymerase II in Southwestern DNA binding assays we have identified regions that have the potential to bind nucleic acids non-specifically. A region exhibiting strong DNA binding is located in the N-terminal part of the molecule (amino acids 357-504) and some weak DNA binding is observed for the C-terminal part (amino acids 860-1160). The non-specific DNA binding behavior of these regions is similar to that of the native enzyme.

Baculovirus expression of the nucleocapsid protein of a Puumala serotype Hantavirus.

Recombinant baculoviruses were generated harboring the entire coding region of the S segment cDNA of Hantavirus strain CG 18-20 that belongs to the Puumala serotype. The recombinant nucleocapsid protein was expressed in Sf9 cells and shown to be antigenically identical with the authentic viral nucleocapsid protein by means of immunoblot analysis. Acute-phase and convalescent sera from European HFRS patients recognized the recombinant nucleocapsid protein in Western blots and the recombinant Baculovirus in indirect immunofluorescence assays.

The gene upstream of DmRP128 codes for a novel GTP-binding protein of Drosophila melanogaster.

Upstream of the gene coding for the second-largest subunit of RNA polymerase III (DmRP128) we have found another gene (128up), which is transcribed in the same direction as the RNA polymerase gene. The intergenic distance between the 3' end of 128up mRNA and the 5' end of DmRP128 mRNA is only about 100 bp. Transcripts of 128up are present at a much higher level than DmRP128 RNA in Drosophila Schneider 2 cells, embryos, and adult flies.

RPII15 codes for the M(r) 15,000 subunit 9 of Drosophila melanogaster RNA polymerase II.

The RPII15 gene product of Drosophila melanogaster, which has recently been identified by sequence comparison, possesses a high similarity to subunit 9 of yeast RNA polymerase II. Using the polymerase chain reaction the coding region of RPII15 was isolated from genomic DNA of adult flies. Sequence analysis shows four amino acid substitutions in comparison to the previously reported sequence.

RNA binding of recombinant nucleocapsid proteins of hantaviruses.

Genes encoding the nucleocapsid (N) proteins of two hantaviruses, Hantaan virus strain 76-118 (HTN) and Puumala virus strain CG 18-20 (PUU), were expressed in Escherichia coli as histidine-tagged proteins. They were purified by metal-chelate affinity chromatography under native or denaturing conditions to near homogeneity. The soluble form of HTN N protein was associated with RNA of E.

A novel mu-capture enzyme-linked immunosorbent assay based on recombinant proteins for sensitive and specific diagnosis of hemorrhagic fever with renal syndrome.

Hantavirus nucleocapsid protein has recently been identified as a major antigen inducing an early and long-lasting humoral immune response in patients with hemorrhagic fever with renal syndrome. A mu-capture enzyme-linked immunosorbent assay utilizing recombinant nucleocapsid proteins of Hantavirus strains Hantaan 76-118 (Hantaan serotype) and CG 18-20 (Puumala serotype) as diagnostic antigens and specific monoclonal antibodies as the detection system has been developed. Histidine-tailed recombinant proteins were expressed in Escherichia coli and purified in a single step by affinity chromatography on a nickel-chelate resin.

Use of recombinant nucleocapsid proteins of the Hantaan and nephropathia epidemica serotypes of Hantaviruses as immunodiagnostic antigens.

Hantavirus nucleocapsid protein has previously been identified as the major antigen recognized by the humoral immune response in hemorrhagic fever with renal syndrome (HFRS). It was therefore considered to be a suitable antigen for the development of rapid and reliable immunodiagnostic assays. Genes encoding the nucleocapsid proteins of two Hantavirus strains, one of the Puumala serotype [nephropathia epidemica virus (NEV)] and the other of the Hantaan serotype were expressed in E.

Identification of a nucleic acid-binding region within the largest subunit of Drosophila melanogaster RNA polymerase II.

The largest and the second-largest subunit of the multisubunit eukaryotic RNA polymerases are involved in interaction with the DNA template and the nascent RNA chain. Using Southwestern DNA-binding techniques and nitrocellulose filter binding assays of bacterially expressed fusion proteins, we have identified a region of the largest, 215-kDa, subunit of Drosophila RNA polymerase II that has the potential to bind nucleic acids nonspecifically. This nucleic acid-binding region is located between amino acid residues 309-384 and is highly conserved within the largest subunits of eukaryotic and bacterial RNA polymerases.