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. The IgG expression cassette elements, including the authentic IgG lambda or kappa and heavy chain signal sequences, as well as light chain (lambda or kappa) and heavy chain constant region genes are combined in a single vector and are controlled by the p10 and polyhedron promoter respectively. Either of VL or Fab-L and VH or Fab-Fd genes from common phage display systems can be directly inserted into one of the cassette vectors through in-frame cloning sites. This design of a single cassette vector combining heavy and light chain expression elements allowed rapid production and secretion of correctly processed and assembled intact immunoglobulins from recombinant baculovirus infected insect cells. The recombinant antibodies showed the expected molecular size of the H2L2 heterodimer in non reducing SDS-PAGE. No apparent differences were found between the expression level of heavy and light chains, and antigen binding function was preserved. For various antibodies, yields between 6 and 18 mg/l IgG were obtained.

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. Both replacements resulted in a total loss of binding activity suggesting that the antigen binding site is contributed by both chains. The functional contributions of each complementarity determining region (CDR) were investigated by site specific mutagenesis of each CDR separately. Mutations in two of the CDRs, CDR1 of light chain and CDR2 of heavy chain, reduced the binding activity significantly. Each of the amino acids in these two CDRs was replaced individually by alanine (alanine walking). Seven amino acid substitutions in the two CDRs were found to reduce the binding activity by more than 50%. The data support a computer model of scFv215 which fits an epitope model based on a mutational analysis of the epitope suggesting an alpha-helical structure for the main contact area.

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. It contains 12 cysteines, and represents parts of the second, the entire third, and parts of the fourth, of the eight clustered 'ligand binding repeats' in LR8; only two of the eight stretches of negatively charged residues of LR8, i.e., EDGSDE and DSGEDEE, are present. The latter sequence is reminiscent of that in the fifth repeat of the human LDLR, thought to be most critical for interaction with positive charge clusters in ligands. Baculovirus-mediated expression of the soluble minireceptor in insect cells showed it to fold as a monomer, and sulfhydryl-reduction-sensitive interaction with RAP was demonstrated for immobilized as well as soluble minireceptor. Furthermore, the LR8-derived minireceptor provided a RAP-responsive surface when covalently coupled to the surface of a gold electrode. In addition to its use in defining minimal binding domains, the phage display approach provides powerful tools for dissection, and consequently, manipulation, of the function of receptors so as to direct their binding activity toward ligands of diagnostic and/or therapeutic interest.

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. In contrast, biopanning of 6mer and 15mer random peptide libraries was only successful for two of the antibodies (mAbBp53-11 and mAbGDO5) after three or four rounds of selection. For the anti-p53 antibody we recovered the epitope from both the 6mer and 15mer library, for mAbGDO5 only the 6mer library displayed the epitope sequence. However, screening of the random peptide libraries with mAb215 and mAbL13F3 failed to yield immunopositive clones. Fine mapping of the epitopes by peptide scan revealed that the minimal epitopes recognized by mAbBp53-11 and mAbGDO5 consist of four and five amino acids, respectively, whereas mAb215 requires a minimal epitope of 11 amino acids for antigen recognition. In contrast, mAbL13F3 did not react with any of the synthesized 15mer peptides. The limits of the different methods of epitope mapping tested in this study are compared and the advantages of the gene-fragment phage display system are discussed.

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. The antigenicity of these proteins was tested in enzyme immunoassays and immunoblots. These studies revealed that human IgG immune response is primarily directed against epitopes located within the amino acid residues 1 to 119 of the amino terminus of viral nucleocapsid proteins. This fragment was recognized by all HFRS patient sera tested (n = 128). The corresponding enzyme immunoassays proved to be more sensitive than the indirect immunofluorescence assays. Furthermore, the majority of bank vole monoclonal antibodies raised against Puumala virus reacted specifically with this site. A recombinant G1 protein (aa 59 to 401) derived from the CG 18-20 strain was recognized by 19 out of 20 sera from HFRS patients.

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. The cDNA of the 100 kDa protein has been cloned recently and its immunogenic regions have been partially mapped using recombinant proteins. Thus far the localization of antigenic determinants on polypeptides has been done by expressing defined cDNA fragments in bacteria or by synthesizing overlapping short peptides and probing their immunoreactivity with antibodies. Here we present an alternative approach to localize autoimmune epitopes using sera containing polyclonal antibodies and gene-fragment phage display libraries. For epitope fine mapping of the PM/Scl-100 protein random fragments of the corresponding cDNA were cloned into the PIII protein of fUSE-5. These gene-fragment phage display libraries were incubated with affinity purified anti-PM/Scl-100 antibodies to enrich for epitope-displaying phages. All PM/Scl sera tested recognized 23 consecutive amino acids (229-251) encoded by four overlapping fUSE-5 clones, suggesting that a major epitope is contained within the 23 amino acids. In addition a minor epitope was localized in a region of 21 amino acids (775-795) encoded by two overlapping fUSE-5 clones since only three out of the seventeen sera reacted with this amino acid sequence. Additional fine mapping of the major epitope was done using synthetic oligopeptides. Thus, a stretch of 16 amino acids at position 229-244 could be identified as a major epitope on the deduced PM/Scl-100 amino acid sequence.

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. The epitopes for the two monoclonal antibodies were mapped using subunit-specific phage display libraries, thus allowing a direct correlation of the structural data with functional information on conserved sequence elements. An epitope close to conserved region C of the beta-like subunit is located at the base of the finger-like domain, whereas a sequence between conserved regions C and D of the beta'-like subunit is located in the apical region of the enzyme. Polyclonal antibodies outlined the alpha-like subunit AC40 and subunit AC19 which were found co-localized also in the apical region of the enzyme. The spatial location of the subunits is correlated with their biological activity and the inhibitory effect of the antibodies.

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. A monoclonal antibody (mAb 215) directed against the largest subunit of Drosophila RNA polymerase II (RPB215) was used to map the corresponding epitope in a fUSE5 phage display library made of random DNA fragments from plasmid DNA containing the entire gene. After a single round of panning with this phage library, bacterial colonies were obtained which produced fd phages displaying the mAb 215 epitope. Sequencing of single-stranded phage DNA from a number of positive colonies (recognized by the antibody on colony immunoblots) resulted in overlapping sequences all containing the 15mer epitope determined by mapping with synthetic peptides. Similarly, we have localized the epitopes recognized by a mouse monoclonal antibody directed against the human p53 protein, and by a mouse monoclonal antibody directed against the human cytokeratin 19 protein. Identification of positive colonies after the panning procedure depends on the detection system used (colony immunoblot or ELISA) and there appear to be some restrictions to the use of linker-encoded amino acids for optimal presentation of epitopes. A comparison with epitope mapping by synthetic peptides shows that the phage display method allows one to map linear epitopes down to a size only slightly larger than the true epitope. In general, our phage display method is faster, easier, and cheaper than the construction of overlapping fusion proteins or the use of synthetic peptides, especially in cases where the antigen is a large polypeptide such as the 215 kDa subunit of eukaryotic RNA polymerase II.

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. The epitope is not involved in the polymerization reaction or the DNA unwinding process since no inhibitory effects of the monoclonal antibody were observed in nonspecific in vitro transcription using denatured calf thymus DNA or double stranded oligo dC-tailed T7 DNA as template. In contrast, MAb215 inhibits accurate in vitro transcription from the Krüppel gene promoter and from the adenovirus-2 major late promoter. Preincubation of template DNA with the nuclear extract had no effects on inhibition supporting the notion that the epitope does not participate directly in the formation of preinitiation complexes. The same inhibitory effects were observed using scFv215. The results provide further evidence that recombinant antibody fragments produced in Escherichia coli possess the same specificity and similar affinity as their parental antibodies and demonstrate that scFv fragments are useful tools for analysis of transcriptional processes.

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. The analysis and the alignment of the nucleotide and the derived amino acid sequences with known sequences of other hantavirus strains demonstrate that Vranica resembles Swedish strains and represents a new virus subtype of the Puumala serotype distinct from the subtypes represented by virus strains CG18-20 and Sotkamo.

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. Most of the known mutations responsible for rifampicin resistance map to a region of the Escherichia coli beta subunit corresponding to the N-terminal nucleic acid-binding region, indirectly supporting the notion that this region participates in interaction with the RNA transcript in ternary complexes.

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. Insect cells infected with the recombinant Baculoviruses proved to be a suitable noninfectious substitute for Hantavirus-infected Vero E6 cells as an antigen source for immunodiagnostic assays allowing the detection of antibodies in HFRS patients.

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. Two transcription start points, seven nucleotides apart, are found for 128up compared to multiple scattered starts for DmRP128. Sequence analysis of 128up cDNA reveals that the gene codes for a 41 kDa protein with homology to GTP-binding proteins and matching four of the structural sequence motifs characteristic of the superfamily of GTPases. Bacterially expressed 128up protein fused to maltose-binding protein specifically binds GTP. Sequences closely related to the 128up protein are found in species as distant as Halobacterium, yeast or mouse; the murine protein is 80% identical to 128up. This evolutionary conservation is indicative of an important, but as yet unknown, physiological role. In accordance with the sequence conservation, antibodies against 128up specifically cross-react with mouse 3T3 cells and human Hep2 cells where the subcellular localization of the protein is predominantly perinuclear. We propose that 128up is a member of a novel class of GTP-binding proteins.

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. Antisera were generated against bacterially expressed RPII15 and were used for immunoblotting experiments with RNA polymerase II of Drosophila melanogaster. This analysis identified the M(r) 15,000 subunit 9 as gene product of RPII15.

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. The assay was evaluated with a panel of sera from patients with hemorrhagic fever with renal syndrome originating from various geographic regions. The overall sensitivity of the mu-capture enzyme-linked immunosorbent assay (both recombinant antigens) was 100%, and its specificity was also found to be 100%. Immunoglobulin M antibodies were detected as early as on day 3, and maximum titers were obtained between days 8 and 25 after onset of the disease. The assay was regularly found to be positive within 3 to 4 months but in some cases up to 2 years after the acute phase of the disease.

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. coli. Renatured N proteins were shown to bind in vitro transcribed RNA representing the hantaviral small genomic (S) RNA segment. RNA binding was shown by affinity to filter-immobilized N proteins and by gel mobility shift assays. Competition experiments using tRNA, poly(U) and poly(A)+ U indicated that binding of RNA by the N protein is nonspecific. However, direct binding of ds-RNA resulted in efficient formation of large complexes suggesting that double-stranded nucleic acids are bound preferentially. Carboxyterminal fragments of HTN and PUU N proteins containing about 100 amino acids of the carboxy termini retained full binding capacity indicating that RNA binding occurs via a carboxyterminal domain.

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. coli, and the expression products were used as diagnostic antigens in solid-phase enzyme immunoassays. The assays were used to detect IgG- and IgM-antibodies in sera of HFRS patients originating from different geographic regions (China, Germany, Greece, Yugoslavia, Scandinavia). ELISA was highly sensitive and proved to be superior to the indirect immunofluorescence assay. Both antigens were necessary to diagnose all HFRS cases originating from the different countries. Most of the sera revealed a predominant reactivity with either 1 of the 2 antigens, allowing the characterization of the etiologic virus as Hantaan-like or NEV-like. The results of the analysis of sera obtained from China and Greece suggested that the Hantaviruses prevalent in these countries are closely related to the Hantaan serotype. In contrast, an NEV-like reactivity was observed in Central and Northern European patients. In the sera of Yugoslav patients both reactivity patterns were found, suggesting that both virus types occur in the Balkan region.

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. A homology to a region of the DNA-binding cleft of Escherichia coli DNA polymerase I involved in binding of the newly synthesized DNA duplex provides indirect evidence that the nucleic acid-binding region of the largest subunit participates in interaction with double-stranded nucleic acids during transcription. The nonspecific DNA-binding behavior of the region is similar to that observed for the native enzyme in nitrocellulose filter binding assays and that of the separated largest subunit in Southwestern assays. A high content of basic amino acid residues is consistent with the electrostatic nature of nonspecific DNA binding by RNA polymerases.

A new approach to the molecular characterization of the c-ANCA antigen in Wegener's granulomatosis.

Classic anti-neutrophil cytoplasmic antibodies (c-ANCA) specific for constituents of neutrophil primary granules and monocyte lysosomes have been implicated in the pathogenesis of Wegener's Granulomatosis (WG). The revised amino-terminal sequence of Proteinase 3 (PR-3) as ANCA antigen, suggested that PR-3 is identical to myeloblastin (MBN). As it has been proposed that autoantibodies recognize a conformational epitope on c-ANCA, prokaryotic expressed protein might not be recognized by the patients sera. Therefore we set up an in vitro translation in an eukaryotic cell-free system using an internal ATG (amino acid 15 of the mature protein).