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Biomedical subjects

H A Küpper

Publications and source records attributed to H A Küpper.

At least 19 recordsLinked to original sources
PubMed1992

Protection of Aotus monkeys from malaria infection by immunization with recombinant hybrid proteins.

On the basis of investigations of the malarial blood-stage antigens SERP, HRPII, and MSAI from Plasmodium falciparum, we chose two Escherichia coli-expressed hybrid proteins containing selected partial sequences of these antigens. Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding P. falciparum polypeptides. In two independent trials with 13 animals, immunization of Aotus monkeys with either of the two hybrid proteins administered in a well-tolerated oil-based formulation protected the animals from an experimental P. falciparum infection.

Animals↗
PubMed1991

Demonstration of alternative splicing of a pre-mRNA expressed in the blood stage form of Plasmodium falciparum.

By screening of a lambda gt11 library from Plasmodium falciparum genomic DNA with an antiserum raised against a 41-kDa protein band, which was shown to confer protective immunity to monkeys, the phage clone 41-3 was identified. The entire 41-3 gene was isolated, and its coding regions were determined by amplification and sequencing of 41-3 specific mRNA fragments. The 41-3 gene has a complex structure consisting of nine exons, encoding 375 amino acids in total with a calculated molecular weight of 43,400. Provided that the N-terminal hydrophobic residues function as signal sequence which is cleaved off, the molecular weight of the 41-3 protein decreases to 41,200 and could therefore be considered to be a component of the protective Mr = 41,000 protein band. Indeed, a 41-kDa protein could be detected by Western blot analysis using antisera raised against different recombinant expression products of the 41-3 gene. We furthermore demonstrate an alternative splice process for the mRNA precursor transcribed from the 41-3 gene to yield at least three distinct mRNAs. The major splice product carries all exons E1 to E9, whereas at least two minor 41-3 mRNA species can be identified which show deletions in the region between exons E5 and E7. The possible role of this differential splice process for the parasite is discussed.

Amino Acid Sequence↗
PubMed1991

Evidence for the location of the receptor-binding site of human erythropoietin at the carboxyl-terminal domain.

Five different peptides (P1: 84-95; P2: 152-166; P3: 52-63; P4: 7-23; P5: 110-123) homologous to relatively hydrophilic regions of human erythropoietin (huEpo) have been synthesized to identify biologically active domains of the hormone. All peptides were able to induce high titers of peptide-specific antibodies in rabbits. Antisera from rabbits induced by recombinant huEpo (rhuEpo) contained a relatively high amount of antibodies preferentially directed against three peptides (P2, P4, and P5), of which P4 comprised the amino-terminal region, P2 the carboxyl-terminus, and P5 an interior region previously described as the receptor-binding site. The same three peptides were able to induce rhuEpo-specific antibodies, whereas P1 and P3 lacked this activity. Only peptide-P2-induced antisera inhibited the biologic activity of rhuEpo in a cell proliferation assay, indicating that the carboxyl-terminal region of the molecule is essentially involved in the biologic function of rhuEpo.

Amino Acid Sequence↗
PubMed1991

A new blood stage antigen of Plasmodium falciparum highly homologous to the serine-stretch protein SERP.

We have isolated the gene coding for a new protein (SERP H), highly homologous to the 113-kDa serine-stretch protein SERP which confers protective immunity to monkeys. The gene consists of four exons interrupted by three short introns located at positions corresponding to those of the SERP gene. Both genes were shown to be linked on chromosome 2 of Plasmodium falciparum suggesting that both originate from a common ancestral gene. Both genes are transcribed in the blood-stage form as 3.8-kb mRNAs with high yield. The deduced amino acid sequence of SERP H is highly homologous to SERP, although it does not contain a serine stretch. A highly hydrophilic region specific for the protein which was shown to be identical among different P. falciparum isolates was expressed in Escherichia coli for preparation of SERP H specific antisera. A schizont polypeptide of 130 kDa within the parasitophorous vacuole was detected by Western blot analysis and immunoelectron microscopy. Like SERP, the 130-kDa protein exhibits a region homologous to cysteine proteinases, suggesting that these proteins, or their processing products, may play a role as proteinases at the time of merozoite release from the infected erythrocyte.

Amino Acid Sequence↗
PubMed1991

Surface expression of malarial antigens in Salmonella typhimurium: induction of serum antibody response upon oral vaccination of mice.

The Escherichia coli OmpA protein can serve as a carrier for the expression of foreign antigens on the surface of gram-negative bacteria. Employing OmpA vectors, immunogenic moieties of the Plasmodium falciparum blood stage antigens SERP and HRPII have been expressed in the attenuated Salmonella typhimurium SR-11 strain. Upon induction, the malaria specific sequences of 189 (HRPII) and 451 (SERP) amino acids, fused into the OmpA protein, have been expressed. By indirect immunofluorescence studies, live bacteria expressing the fusion proteins react anti-SERP and anti-HRPII sera, respectively, indicating that the hybrid OmpA proteins become integrated into the bacterial outer membrane and expose the malarial antigens at the exterior surface. Mice that were immunized orally with S. typhimurium cells expressing HRPII and SERP on their surface show a humoral immune response as determined by the anti-SERP and anti-HRPII IgG and IgM titres. From these experiments it can be concluded that the OmpA surface expression system in combination with established Salmonella vaccine strains can be used to efficiently deliver large antigens to the mucosal immune system.

Administration, Oral↗
PubMed1991

A recombinant hybrid protein as antigen for an anti-blood stage malaria vaccine.

Based on investigations on several blood stage antigens from Plasmodium falciparum we have expressed a hybrid protein in E. coli containing 262 amino acids of the serine-stretch protein SERP and 189 amino acids of the histidine alanine rich protein HRPII. Antibodies raised against the hybrid protein by immunization of rabbits and Aotus monkeys react with both corresponding schizont polypeptides. Two Aotus monkeys immunized with the SERP/HRPII hybrid protein showed only low parasitemias after challenge infection with P. falciparum, compared to the control group. The result suggests that hybrid proteins of this type may be the basis for the development of a malaria vaccine.

Animals↗
PubMed1990

Plasmodium falciparum aldolase: gene structure and localization.

A genomic clone was isolated which codes for the fructose bisphosphate aldolase of Plasmodium falciparum. The aldolase gene is interrupted by one intron which divides the coding region into two exons. The first one codes for one amino acid only, the initiation methionine, while the second one encodes the residual 368 amino acids of the protein. The gene, which is represented only once in the genome, is transcribed at high rates as a 2.4-kb mRNA in the P. falciparum blood stage. The aldolase gene encodes a protein of 40,105 Da, which is 61-68% homologous to known eukaryotic aldolases. The protein was expressed in Escherichia coli cells in an unfused and enzymatically active form. Antisera raised against amino acids 9-96 recognize a 41-kDa protein band previously shown to protect monkeys against a P. falciparum infection. These antisera cross-react with aldolases of different species, which confirms the strong conservation of this enzyme during evolution. The aldolase could be localized in the cytoplasm of the parasite as an active and soluble form. An inactive form was found to be associated with the membrane fraction. Digestion data with phospholipase C suggest a membrane association of this polypeptide via a glycosylphosphatidylinositol anchor.

Amino Acid Sequence↗
PubMed1990

Characterization of recombinant factor XIIIa produced in Saccharomyces cerevisiae.

Recombinant factor XIIIa (FXIIIa), produced in Saccharomyces cerevisiae, was recovered as a fully active cytosolic component and rigorously compared to natural F XIIIa from human placenta with respect to physicochemical and functional properties. Identical parameters were found in SDS polyacrylamide gel electrophoresis, analytical ultracentrifugation and HPLC gel filtration, and all spectral characteristics including derivative UV absorbance, fluorescence and circular dichroism were identical. Similarly, the interaction of both proteins with polyclonal antibodies directed against the entire FXIIIa or its N-terminal 4 kD activation peptide were identical. Furthermore, thrombin cleavage and fibrin cross-linking showed indistinguishable patterns. The only difference we observed was with respect to endgroup analysis. The recombinant protein is homogeneous, whereas placental FXIIIa shows multiple electrophoretic bands caused by microheterogeneity in the C-terminal part of the protein.

Cloning, Molecular↗
PubMed1989

Molecular cloning, genomic structure and localization in a blood stage antigen of Plasmodium falciparum characterized by a serine stretch.

Two short DNA segments were isolated by screening of a lambda gt11 library from Plasmodium falciparum schizont cDNA with an antiserum against the 140 kDa protein, which confers protective immunity to monkeys. The segments were used to identify a genomic fragment which carries the entire coding sequence for a protein of 113 kDa characterized by a stretch of serine residues (SERP I). We present the complete nucleotide and deduced amino acid sequence as well as the structure of the SERP I gene. The gene consists of four exons interrupted by three short introns located at the amino-terminal half. Exon 1 and the first part of exon 2 code for hydrophobic amino acids of a putative signal sequence. Exon 2 contains two repetitive segments, the first encoding six glycine rich octapeptides and a second region coding for 37 consecutive serine residues. Southern blot analysis demonstrated the conservation of the SERP I gene in four different parasite strains. SERP I could be localized in the parasitophorous vacuole and in the surrounding membranes. We discuss the relationship of this protein to the recently described P126 polypeptide and the possible role of this antigen as a vaccine candidate.

Amino Acid Sequence↗
PubMed1989

A new blood stage antigen of Plasmodium falciparum transported to the erythrocyte surface.

On screening a lambda gt11 library from Plasmodium falciparum genomic DNA with an antiserum against the 41-kDa protein band, which confers protective immunity to monkeys, two strongly reacting clones were isolated. One of the clones codes for parts of the P. falciparum 41-kDa aldolase, while the other (41-2) codes for parts of an unknown antigen; this was analyzed further. The 41-2 insert was used to identify two genomic fragments which carry the entire gene. The 41-2 gene codes for 184 amino acids specifying a 29-kDa schizont protein as estimated by Western blot analysis. The protein contains no repetitive sequences, and is characterized by a signal sequence and by two additional hydrophobic segments which could function as membrane anchor sequences. Computer analysis of the protein sequence predicted a dominant epitope in the N-terminal part which was confirmed by immunoreactivity data. The 41-2 protein could be localized in the schizont membrane, associated with membranous structures in the erythrocytic cytoplasm and with the erythrocyte membrane.

Animals↗
PubMed1988

Synthesis of human factor XIIIa in bacterial cells.

The coding sequence for human factor XIIIa (FXIIIa) was introduced into Escherichia coli expression vectors. Bacterial cells transformed with the recombinant plasmids synthesized fusion proteins of the expected molecular weights and the proteins were shown to be immunoreactive with anti-FXIII antibodies. Furthermore, with the help of oligodeoxynucleotide synthesis, we constructed a plasmid which directs the synthesis of the human FXIIIa protein in the unfused form. Sequence determination at the aminoterminus of this protein revealed the identical sequence compared to placental FXIIIa. The protein is expressed intracellularly in a denatured and biologically inactive form. It constitutes approximately 2% of total cellular protein and can easily be purified by standard methods.

Amino Acid Sequence↗
PubMed1988

Isolation and expression of cDNA coding for a new member of the phospholipase A2 inhibitor family.

The placental protein PP41,2 was shown to have thromboplastin-inhibitor activity. We used partial amino acid sequence information from PP4 cyanogen bromide fragments to design oligonucleotide probes for the screening of a human placental cDNA library. In addition to the PP4 cDNA we isolated a cDNA coding for a protein with considerable homology which we subsequently termed PP4-X. PP4 and PP4-X belong to the phospholipase A2 inhibitor family, as judged by their homology to lipocortin I and calpactin I3. The full-length PP4-X cDNA encodes a protein of 321 amino acid residues including a fourfold repeat structure. Northern blot analysis using the PP4-X cDNA reveals two hybridizing RNA species of approximately 1400 nucleotides and 2500 nucleotides, respectively. The shorter one could well represent the PP4-X transcript which is in good agreement with the isolated cDNA insert of 1326 nucleotides. Expression of the PP4-X coding sequence in E. coli resulted in the appearance of a protein which crossreacts with antibodies raised against PP4.

Annexin A5↗
PubMed1988

Efficient expression system for human antithrombin III in baby hamster kidney cells.

A DNA fragment carrying the enhancer of an immediate early gene of human cytomegalovirus (hCMV) was tested as a transcriptional control element by fusion downstream of a transcription unit for human antithrombin III (AT III) driven by the Simian virus (SV40) enhancer and promoter. Measurement of transient AT III expression in baby hamster kidney cells (BHK) shows that by the presence of the hCMV enhancer the synthesis of AT III is increased considerably (three to fourfold). The AT III expression vectors carrying the hCMV enhancer were used to establish stable BHK cell-lines by a new and fast G418/methotrexate selection protocol, which express up to 12 micrograms AT III/10(6) cells/24h after 40-50 days. The given system might be generally useful for the fast expression of recombinant proteins in mammalian cells, e.g. the screening of altered AT III molecules obtained by site directed mutagenesis.

Animals↗
PubMed1988

Immunoreactivity of human immunodeficiency virus (HIV-1) envelope polypeptides expressed in Escherichia coli.

Defined cDNA sequences encoding segments of envelope glycoproteins of the human immunodeficiency virus were expressed in E. coli. The recombinant env fusion proteins were tested for immunoreactivity with patient sera by radio immunoprecipitation procedures. The regions of the env proteins, carrying relevant antigenic determinants in respect to diagnostic purposes, could be identified.

Cloning, Molecular↗
PubMed1988

A histidin alanine rich recombinant antigen protects Aotus monkeys from P. falciparum infection.

We have isolated a cDNA clone coding for 165 amino acids of a histidine alanine rich protein. An extended repeat region of this clone codes for the tripeptide Ala-His-His, which is extremely conserved, and for the tripeptide Ala-Ala-Asp, which shows only slight variability among the repeat units. This antigen exhibits high homology to the HRPII antigen described by Wellems and Howard (1986). The coding region was expressed in E. coli as a MS2-polymerase fusion protein, which was purified and used for immunization of Aotus monkeys. The animals immunized with this fusion protein showed only low parasitemias (less than 2%) after infection with P. falciparum, while animals from the control group or animals immunized with a MS2-fusion protein carrying other malaria specific sequences were not protected. The result suggests that this antigen is a good candidate for a malaria vaccine.

Amino Acid Sequence↗
PubMed1988

Synthesis of biologically active deletion mutants of human factor VIII:C.

The recent cloning and sequence analysis of human factor VIII:C (antihaemophilic factor) revealed a domain structure for the protein which can be presented as A1-A2-B-A3-C1-C2. In this report we describe the construction of two altered factor VIII:C cDNAs coding for molecules in which a part (amino acids 816 to 1598) or all of the B domain (amino acids 741 to 1689) was removed. In the latter mutant a new thrombin cleavage site has been created, which does not exist in wild-type factor VIII:C. The mutated cDNAs were cloned into eucaryotic expression vectors based on regulatory sequences of the virus SV40 and transfected into two different mammalian cell lines. Both truncated recombinant factor VIII:C molecules were secreted into the culture medium, showed full biological activity and could be activated by thrombin.

Amino Acid Sequence↗
PubMed1986

Characterization of cDNA coding for human factor XIIIa.

A cDNA library prepared from human placenta has been screened for sequences coding for factor XIIIa, the enzymatically active subunit of the factor XIII complex that stabilizes blood clots through crosslinking of fibrin molecules. Two oligonucleotides, based on the amino acid sequences of tryptic peptides of factor XIIIa, were used as hybridization probes. Of 0.36 X 10(6) independent recombinants, 1 clone was identified that hybridized to both probes. The insert of 1704 base pairs coded for the amino-terminal 541 amino acid residues of the mature factor XIIIa molecule. Blot-hybridization analysis using this cDNA as a probe showed that the factor XIIIa mRNA from placenta has a size of approximately 4000 bases. The insert was used to rescreen cDNA libraries and to identify further factor XIIIa-specific sequences. The total length of the isolated factor XIIIa cDNA is 3905 bases, and it codes for a protein of 732 amino acids. In spite of the presence of factor XIII in blood plasma, we could not identify a leader sequence typical for secreted proteins.

Amino Acid Sequence↗