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Biomedical subjects

E Amann

Publications and source records attributed to E Amann.

At least 37 records · Page 2Linked to original sources
PubMed1991

Expression of the human blood coagulation protein factor XIIIa in Saccharomyces cerevisiae: dependence of the expression levels from host-vector systems and medium conditions.

The human blood coagulation protein Factor XIIIa (FXIIIa) was expressed in Saccharomyces cerevisiae employing Escherichia coli-yeast shuttle vectors based on a 2-mu plasmid. Several factors affecting high production yield of recombinant FXIIIa were analysed. The use of the regulatable GAL-CYC1 hybrid promoter resulted in higher FXIIIa expression when compared with the constitutive ADCI promoter. Screening for suitable yeast strains for expression of FXIIIa under the transcriptional control of the GAL-CYC1 hybrid promoter revealed a broad spectrum of productivity. No obvious correlation between the expression rate and the genetic markers of the strains could be identified. The medium composition markedly influenced the FXIIIa expression rates. The expression of FXIIIa was strictly regulated by the carbon source. Glucose as the only sugar and energy source repressed the synthesis of FXIIIa, whereas addition of galactose induced FXIIIa expression. Special feeding schemes resulted in a productivity of up to 100 mg FXIIIa/l in shake flasks.

Chromosomes, Fungal↗
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↗
PubMed1990

Annexin proteins PP4 and PP4-X. Comparative characterization of biological activities of placental and recombinant proteins.

The human placental proteins PP4 and PP4-X, belonging to the annexin protein family, were expressed in Escherichia coli at high yield. The proteins were purified to homogeneity. The physicochemical parameters of the recombinant proteins were determined and compared with those of their natural placental counterparts. Except for a minor change in the pI, the proteins appeared to be indistinguishable by several criteria. Both recombinant PP4 and recombinant PP4-X were biologically active in a thromboplastin inhibition test and in a phospholipase A2 inhibition test.

Annexin A5↗
PubMed1990

Escherichia coli-derived envelope protein gD but not gC antigens of herpes simplex virus protect mice against a lethal challenge with HSV-1 and HSV-2.

Immunization studies with HSV-1 and HSV-2 envelope proteins expressed in Escherichia coli were performed. After active immunization of mice with a gD-1 antigen (Leu53-Ala312) expressed as a fusion protein, the animals were protected from a lethal challenge with HSV-1 and HSV-2. In addition, antisera from rabbits immunized with the same gD-1 antigen also conferred passive immunity to mice against a challenge infection with either HSV-1 or HSV-2. In contrast to these successful gD-1 protection experiments, various gC-1 and gC-2 fusion proteins from E. coli failed to induce protective immunity. Moreover, the mice sera from immunized animals were not able to react with the authentic, glycosylated gC-1 and gC-2 envelope proteins, whereas sera raised against authentic gC-1 and gC-2 glycoproteins do recognize the gC fusion proteins from E. coli. These results indicate, that E. coli might represent an ideal system for expressing gD antigens as a possible component of a HSV vaccine, whereas gC antigen cannot be produced in an immunocompetent form in E. coli.

Animals↗
PubMed1990

Cloning and prokaryotic expression of a biologically active human placental aldose reductase.

cDNA clones coding for human aldose reductase (AR) were isolated by antibody screening of a placental lambda gt11 cDNA library. The cDNA comprises the entire coding region and has a total length of 1,394 bp. The sequence deduced from the open reading frame encodes a protein of 316 amino acids and its amino acid composition is identical to the placental protein 9 (PP9), whose isolation and characterization were described by Bohn et al. (1982). The amino acid sequence of the placental human AR shows high homology to the rat AR; both proteins belong to the same protein superfamily as human liver AR, frog lens rho-crystallin, and bovine lung prostaglandin F synthase. Northern blot hybridization analysis revealed a size for the AR mRNA of approximately 1,500 bases. In addition to the full-length cDNA, one lambda gt11 clone was isolated which carries a putative intron of 597 bp at nucleotide position 754, corresponding to amino acid position 247. Expression of the AR cDNA in Escherichia coli resulted in the synthesis of a protein with a molecular weight of approximately 35 kD which can be immunoprecipitated specifically with antiserum raised against PP9. Despite the absence of a typical signal sequence, the human aldose reductase is partially translocated into the periplasm of the E. coli cells, where it is present in an enzymatically active form.

Aldehyde Reductase↗
PubMed1990

Cloning and expression of a cDNA encoding human placental protein 11, a putative serine protease with diagnostic significance as a tumor marker.

The placental protein 11 (PP11) can act as a tumor marker because of its specific association with various forms of cancer. A lambda gt11 cDNA library prepared from human placenta was screened with a polyclonal anti-PP11 antiserum. Out of 10(6) independent clones, only one clone reacted with the anti-PP11 antiserum. The isolated cDNA coded only for the carboxy-terminal part of PP11 and was subsequently used to rescreen a lambda gt10 placental cDNA library. Two cDNA clones out of 10(6) screened were identified encoding the entire protein of 369 amino acids, including a typical hydrophobic signal sequence of 18 amino acids. Expression of the PP11 cDNA coding sequence in Escherichia coli resulted in the synthesis of a protein with the expected size which can be specifically immunoprecipitated with anti-PP11 antiserum. Fractionation experiments revealed that two forms of the protein are present in the bacterial cell: a higher-molecular-weight form of approximately 42 kD in the cytoplasm and a smaller-molecular-weight form of approximately 42 kD in the periplasm. This result indicates that PP11 can be synthesized in E. coli and is process by removal of the hydrophobic signal sequence. Both the placental and the processed recombinant PP11 protein exhibit a protease activity.

Amino Acid Sequence↗
PubMed1988

Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli.

A series of new plasmid expression vectors (the pTrc series) has been constructed for the regulated expression of genes in Escherichia coli. Based on pKK233-2 [Amann and Brosius, Gene 40 (1985) 183-190], the vectors carry a strong hybrid trp/lac promoter, the lacZ ribosome-binding site (RBS), the multiple cloning site of pUC18 and the rrnB transcription terminators. With the aid of synthetic oligodeoxynucleotides, the multiple cloning site has been inserted behind an NcoI site in three reading frames. Thus, the vectors are equally useful for the expression of proteins in their authentic, non-fused form (by using the NcoI site) and for the expression of fusion proteins (by choosing any of the cloning sites in the correct translational frame). To ensure complete repression of the hybrid trp/lac promoter during construction and growth in any host strain, the lacIq allele of the lac repressor gene was added to some of the vectors. The complete vector nucleotide sequence and examples of heterologous gene expression (human coagulation factor XIIIa and human placental anticoagulant protein PP4) with the new vectors are presented.

Bacterial Proteins↗
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

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↗
PubMed1985

"ATG vectors' for regulated high-level expression of cloned genes in Escherichia coli.

A plasmid cloning vector system has been constructed that allows for the production of large quantities of foreign proteins or fragments thereof, in an unfused state. These vectors provide strong regulated trp-lac fusion promoters and the lacZ ribosome-binding site (RBS) followed by an ATG translation initiation codon at an appropriate distance from the RBS. The ATG codon is located within a unique NcoI restriction site (CCATGG). Digestion with NcoI exposes the ATG for fusion. Gene fragments lacking a prokaryotic RBS and/or ATG start codons can be inserted in several ways. Expression experiments using a truncated cI gene of bacteriophage lambda or a large portion of the coding region of the Herpes simplex virus type 1 glycoprotein D gene have been performed. The results of these studies show that the vectors are useful for the high-level expression of prokaryotic and eukaryotic genes in Escherichia coli.

Base Sequence↗
PubMed1985

Plasmid vectors for the regulated, high level expression of eukaryotic genes in Escherichia coli.

A series of plasmid vectors has been constructed for the regulated, high level expression of foreign genes in E. coli. The vectors express cloned genes under the control of the tac promoter, which is a hybrid of trp and lac promoter sequences. Some of our expression vectors carry in addition to the tac promoter, the efficient lacZ ribosome binding site followed by unique cloning sites. These vectors can be used to express cloned genes directly, i.e. in an unfused, mature form. A second type of vector provides, in addition to the above regulatory elements, a translation initiation codon (ATG) for the expression of genes which have been isolated in an incomplete form (for example: cDNA). A third type of vector allow readily the construction of gene fusions to the E. coli beta-galactosidase gene, which may stabilize otherwise unstable eukaryotic proteins, and thus allows the production of high amounts of specific antigens in E. coli. With the above vectors, several eukaryotic and viral proteins, including SV40 small tumor antigen, human fibroblast interferon and herpes simplex glycoproteins have been expressed.

Antigens, Polyomavirus Transforming↗
PubMed1984

Ventricular arrhythmias before and late after aortic valve replacement.

The influence of aortic valve replacement on the incidence of ventricular arrhythmias was studied by 24-hour Holter electrocardiographic monitoring in 45 patients immediately before and 14 +/- 7 months after operation. Ventricular arrhythmias were graded according to the Lown criteria. Preoperative left ventricular (LV) ejection fraction (EF) was determined by angiography and postoperative LVEF by gated blood pool scintigraphy. Repetitive ventricular arrhythmias (Lown grade 4A/B) were associated with a reduced LVEF (less than 55%) before and after operation. In 24 patients with preoperative normal LVEF (greater than or equal to 55%) (group A), mean LVEF remained unchanged after operation (72% vs 71%). Pre- and postoperative ventricular premature complex (VPC) frequency (45 +/- 99 vs 39 +/- 94 VPC/24 hours) and grade (1.3 vs 1.4) were not significantly different. However, in 17 patients with preoperative impaired LVEF (less than 55%) (group B, LVEF preoperatively 40 +/- 8%) and marked postoperative improvement (greater than 10%) (LVEF postoperatively 64 +/- 7%), mean VPC frequency decreased from 536 to 69 VPCs/24 hours and mean VPC grade was reduced from 3.8 to 1.5. Complex VPCs were found preoperatively in all 17 patients of group B, but in only 5 patients after operation. Four patients had a reduced LVEF preoperatively and it did not improve postoperatively (group C). Postoperative Holter monitoring detected ventricular tachycardia in all 4 patients. This study indicates that repetitive VPCs are infrequent in patients with normal LVEF before and late after aortic valve replacement. In patients with impaired LVEF and complex VPCs preoperatively, the postoperative improvement of LV function is usually accompanied by a reduction of frequent and complex VPCs.

Adolescent↗
PubMed1984

Expression of Herpes simplex virus type 1 glycoprotein C antigens in Escherichia coli.

DNA fragments encoding structural information of the Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) gene were cloned into pUC plasmids [Vieira and Messing, Gene 19 (1982) 259-268]. None of the hybrid plasmids were able to direct the synthesis of significant amounts of gC related peptides. Several of the plasmid-bearing strains, however, exhibited inhibition characteristics which can be correlated with the presence on the plasmid of specific gC gene sequences. After insertion of gC DNA fragments into expression vector pMF2 between phage lambda repressor gene cI and lacZ, significant amounts of cI::gC::beta-galactosidase fusion proteins are synthesized. These tripartite fusion proteins are immunologically reactive with anti-HSV-1 antisera. The expression system based on pMF2 can be generally used to identify and express foreign antigens in Escherichia coli.

Antigens, Viral↗
PubMed1983

Lack of interaction between the tetracyclic antidepressant maprotiline and the centrally acting antihypertensive drug clonidine.

The well known interaction between tricyclic antidepressants and the centrally acting antihypertensive drug clonidine, namely impairment of the antihypertensive effect of clonidine, is thought to be related to blockade of noradrenaline uptake or competition at central alpha-receptors. The tetracyclic antidepressant maprotiline has been shown to be a potent inhibitor of noradrenaline uptake and it might, therefore, interfere with the antihypertensive action of clonidine. The possible interaction of clonidine and maprotiline was studied in 8 healthy subjects using doses in the therapeutic range. The study followed a double-blind, cross over design, in which clonidine alone (0.3 mg p.o.), clonidine (0.3 mg p.o.) plus maprotiline (100 mg in 4 divided doses over 22 h), maprotiline alone (100 mg in 4 divided doses over 22 h) and placebo were given by the double-dummy technique. Several pharmacodynamic parameters were measured for 12 h after administration of the drugs (supine and erect blood pressure, heart rate, saliva production and sedation). Concurrent administration of maprotiline did not alter the effect of clonidine and neither the size nor the time of the maximal response after clonidine were influenced by maprotiline. It is concluded that [1] blockade of noradrenaline uptake is not associated with the interaction of tricyclic antidepressants and clonidine, and [2] maprotiline should be preferred to tricyclic antidepressants in hypertensive patients on clonidine therapy if a concomitant depressive illness has to be treated.

Adult↗
PubMed1983

Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors.

A broad-host-range vector, pKT240, containing the structural gene (aph) for aminoglycoside phosphotransferase (APH), without promoter, has been constructed. Insertion of DNA fragments carrying promoters upstream of aph gene into the unique EcoRI site of this vector results in the expression of the aph gene and consequently the resistance of the host cells to streptomycin. The new vector has been used to show that the hybrid trp-lac (tac) promoter and the promoter of the lacIQ gene of Escherichia coli are active in Pseudomonas putida. Derivatives of pKT240 containing tac and lacIQ sequences may be used as wide-host-range expression vectors. Regulated overproduction of APH and catechol 2,3-oxygenase can be obtained with the aid of the new vectors in both E. coli and P. putida.

Catechol 2,3-Dioxygenase↗