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A Honegger

Publications and source records attributed to A Honegger.

At least 37 records · Page 2Linked to original sources
PubMed1997

Regulation of the flavin redox potential by flavin-binding antibodies.

Single-chain Fv antibody fragments binding different flavin forms [10-(5'-carboxybutyl-)flavin (Fl[ox]) and 10-(5'-carboxybutyl)-1,5-dihydroflavin (Fl[red])] have been generated from an antibody phage-display library to study how a protein environment regulates the redox potential, starting from a protein other than a natural flavoprotein. These 'flavobodies' are characterized by time-resolved and steady-state fluorescence spectroscopy, by competitive ELISA methods (mapping of the antigen-binding site), and by molecular modelling. The three-dimensional models of the antigen-binding sites are consistent with the experimental results. Binding of anti-Fl(red) 5 to flavin increases the redox potential, mainly due to an Arg residue interacting with the flavin N1. Thus anti-Fl(red) 5 shows an 'oxidase-like' redox-potential behaviour, confirming the idea that positively charged residues in the vicinity of N1 increase the redox potential. The results obtained with anti-Fl(ox), which do not resemble a natural flavoprotein, show that when the pyrimidine-like nucleus of the flavin is not involved in binding, the redox potential is not significantly affected. These results are in contrast to those obtained with chicken riboflavin-binding protein.

Amino Acid Sequence↗
PubMed1997

Affinity and folding properties both influence the selection of antibodies with the selectively infective phage (SIP) methodology.

We investigated which molecules are selected from a model library by the selectively infective phage (SIP) methodology. As a model system, we used the fluorescein binding single-chain Fv fragment FITC-E2, and from a 3D-model, we identified 11 residues potentially involved in hapten binding and mutated them individually to alanines. The binding constant of each mutant was determined by fluorescence titration, and each mutant was tested individually as well as in competitive SIP experiments for infectivity. After three rounds of SIP, only molecules with KD values within a factor of 2 of the tightest binder remain, and among those, a mutant no longer carrying an unnecessary exposed tryptophan residue is preferentially selected. SIP is shown to select for the best overall properties of the displayed molecules, including folding behavior, stability and affinity.

Antibodies↗
PubMed1997

Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.

A prerequisite for the use of recombinant antibody technologies starting from hybridomas or immune repertoires is the reliable cloning of functional immunoglobulin genes. For this purpose, a standard phage display system was optimized for robustness, vector stability, tight control of scFv-delta geneIII expression, primer usage for PCR amplification of variable region genes, scFv assembly strategy and subsequent directional cloning using a single rare cutting restriction enzyme. This integrated cloning, screening and selection system allowed us to rapidly obtain antigen binding scFvs derived from spleen-cell repertoires of mice immunized with ampicillin as well as from all hybridoma cell lines tested to date. As representative examples, cloning of monoclonal antibodies against a his tag, leucine zippers, the tumor marker EGP-2 and the insecticide DDT is presented. Several hybridomas whose genes could not be cloned in previous experimental setups, but were successfully obtained with the present system, expressed high amounts of aberrant heavy and light chain mRNAs, which were amplified by PCR and greatly exceeded the amount of binding antibody sequences. These contaminating variable region genes were successfully eliminated by employing the optimized phage display system, thus avoiding time consuming sequencing of non-binding scFv genes. To maximize soluble expression of functional scFvs subsequent to cloning, a compatible vector series to simplify modification, detection, multimerization and rapid purification of recombinant antibody fragments was constructed.

Amino Acid Sequence↗
PubMed1997

A natural antibody missing a cysteine in VH: consequences for thermodynamic stability and folding.

While the disulfide bridge is highly conserved within the immunoglobulin fold, a few antibody variable domains lack one of the essential cysteine residues. In the levan binding antibody ABPC48 one of the essential cysteine residues (Cys H92) of the heavy chain variable domain is replaced by tyrosine. We expressed scFv fragments with the ABPC48 sequence and a mutant in which the VH disulfide bond has been restored in Escherichia coli, purified both proteins by antigen affinity chromatography and characterized them by equilibrium denaturation. While the ABPC48 protein was found to be significantly less stable than an average scFv molecule, the restored disulfide increased its stability above that of other, unrelated scFv fragments, explaining why it tolerates the disulfide loss. Surprisingly, we observed that under some refolding conditions, the unpaired cysteine residue of functional scFv of ABPC48 is derivatized by glutathione. It is easily accessible to other reagents and thus appears to be solvent-exposed, in contrast to the deeply buried disulfide of ordinary variable domains. This implies a very unusual conformation of stand b containing the unpaired Cys H22, which might be stabilized by interactions with the tyrosine residue in position H92.

Cloning, Molecular↗
PubMed1997

Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment.

By constructing Fv and single-chain Fv (scFv) fragments of antibodies, the variable domains are taken out of their natural context in the Fab fragment, where they are associated with the constant domains of the light (CL) and heavy chain (CH1). As a consequence, all residues of the former variable/constant domain interface become solvent exposed. In an analysis of 30 non-redundant Fab structures it was found that at the former variable/constant domain interface of the Fv fragment the frequency of exposed hydrophobic residues is much higher than in the rest of the Fv fragment surface. We investigated the importance of these residues for different properties such as folding in vivo and in vitro, thermodynamic stability, solubility of the native protein and antigen affinity. The experimental model system was the scFv fragment of the anti-fluorescein antibody 4-4-20, of which only 2% is native when expressed in the periplasm of Escherichia coli. To improve its in vivo folding, a mutagenesis study of three newly exposed interfacial residues in various combinations was carried out. The replacement of one of the residues (V84D in VH) led to a 25-fold increase of the functional periplasmic expression yield of the scFv fragment of the antibody 4-4-20. With the purified scFv fragment it was shown that the thermodynamic stability and the antigen binding constant are not influenced by these mutations, but the rate of the thermally induced aggregation reaction is decreased. Only a minor effect on the solubility of the native protein was observed, demonstrating that the mutations prevent aggregation during folding and not of the native protein. Since the construction of all scFv fragments leads to the exposure of these residues at the former variable/constant domain interface, this strategy should be generally applicable for improving the in vivo folding of scFv fragments and, by analogy, also the in vivo folding of other engineered protein domains.

Amino Acid Sequence↗
PubMed1997

Specific detection of his-tagged proteins with recombinant anti-His tag scFv-phosphatase or scFv-phage fusions.

Using a cell-bound immunogen, we have generated a monoclonal antibody, 3D5, that recognizes carboxy-terminal oligo-histidine tags (His tags) on a wide variety of proteins. From this monoclonal antibody, we have generated a single-chain fragment of the variable domains (scFv), a dimeric scFv-alkaline phosphatase fusion and an oligovalent scFv-display phage. The antibody in its various formats is an effective tool used in fluorescence-activated cell sorting analysis, the BIAcore method, Western blots and enzyme-linked immunosorbent assay (ELISA). Western blots and ELISAs can be developed directly by using crude extracts of E.coli cells that produce the scFv-alkaline phosphatase fusion, thus providing an inexhaustable and convenient supply of detection reagent. Alternatively, oligovalent scFv-displaying phage can be used directly from culture supernatants for this purpose. The dissociation constants, KD of the peptide KGGHHHHH (KD = 4 x 10(-7) M) and of imidazole (KD = 4 x 10(-4) M) were determined. Molecular modeling of the Fv fragment suggests the occurrence of two salt bridges between the protonated histidine side chains of the peptide and the acidic groups in the antibody, explaining why the antibody or the substrate may be eluted under mildly basic conditions.

Antibodies, Monoclonal↗
PubMed1996

Folding nuclei of the scFv fragment of an antibody.

The folding kinetics of the variable domains of the phosphorylcholine-binding antibody McPC603, combined into a scFv fragment [VH-(Gly4Ser)3-VL], were investigated by the use of fluorescence spectroscopy, nuclear magnetic resonance (NMR), and mass spectrometry (MS). All three methods gave evidence for the occurrence of a major kinetic intermediate during the refolding of the denatured, oxidized scFv fragment. This intermediate is formed within the first 30 s of folding and comprises exchange-protected amide protons of hydrophobic and aromatic amino acids, most of which are localized within the inner beta-sheet of the V(L) domain. In the subsequent slow step, most of the amide protons become protected with rate constants that are very similar for residues of both domains. These data are in agreement with the MS results, which indicate a cooperative folding event from the intermediate to the native state of the scFv fragment.

Amino Acid Sequence↗
PubMed1993

Aggregation-induced activation of the epidermal growth factor receptor protein tyrosine kinase.

Various agents are able to stimulate the EGF receptor protein tyrosine kinase in the absence of ligand binding. To characterize their mechanism of action, we investigated their effects on the kinase activity of the intracellular domain of the EGF receptor (EGFR-IC). EGFR-IC (67 kDa) lacking the extracellular domain and transmembrane segment of the EGF receptor, but retaining kinase and autophosphorylation domains, was produced and purified as a soluble, cytoplasmic protein from Sf9 insect cells infected with a recombinant baculovirus. EGFR-IC was able to undergo autophosphorylation in a manner similar to full-length EGFR. Synthetic substrate peptides showed similar affinity to EGFR-IC as to the full-length receptor. The activity of the EGFR-IC was found to be dependent on divalent cations, Mn2+ being a more potent activator than Mg2+. Agents capable of aggregating the kinase by direct interaction (cross-linking antibodies, polycations) or through altering the surrounding solvent structure and thereby decreasing protein solubility [ammonium sulfate, poly(ethylene glycol), 2-methyl-2,4-pentanediol] activated the kinase in a manner which correlated with their ability to precipitate the EGFR intracellular domain. The widely different chemical nature of these agents suggests that they do not act by direct interaction with specific allosteric regulatory sites, but rather by facilitating the interactions between kinase molecules. These results support the hypothesis that full-length receptor aggregation itself, induced by ligand binding to the extracellular domain, results in intracellular domain interactions and the activation of kinase activity.

Animals↗
PubMed1990

Expression of a human insulin-like growth factor II cDNA in NIH-3T3 cells.

Recombinant human insulin-like growth factor II (IGF-II) was produced in NIH-3T3 cells transfected with a plasmid containing a construct encoding the signal peptide and the sequence of mature human IGF II. Successfully transfected clones secreted correctly processed recombinant human IGF II at rates of about 100ng per 24 hours per 10(6) cells. The biological activity of the purified recombinant human IGF II exhibited similar potencies as standard human IGF II isolated from serum in radio-receptorassays as well as in thymidine incorporation assays.

Animals↗
PubMed1990

Generation of recombinant cytoplasmic domain of epidermal growth factor receptor with intrinsic protein tyrosine kinase activity.

We have generated a recombinant baculovirus using the high expression vector pVL941 containing the complementary DNA encoding the intracellular domain of the human epidermal growth factor receptor (EGFR-IC). Upon infection of Spodoptera frugiperda insect cells, protein tyrosine kinase-active EGFR-IC was produced. The expressed protein has a molecular weight of 61,000 and is specifically recognized by antibodies directed against peptides representing different regions of human EGFR-IC. Upon sonication of infected cells, EGFR-IC was detected in both the soluble and insoluble fractions of the cell lysate. About 20-50% of the expressed EGFR-IC was soluble. Metabolic labeling and protein analyses showed that EGFR-IC comprised 7% of newly synthesized proteins in the cytoplasmic lysate and 0.1-0.2% of the total soluble protein. We have used a three-step purification procedure (fast-Q-Sepharose and phenyl-Sepharose column chromatographies and 30% ammonium sulfate precipitation) to purify EGFR-IC to 85% purity with 15-20% recovery from the initial soluble lysate. A yield of 3-4 mg of purified EGFR-IC has been consistently produced from 20 roller bottles with 2-4 x 10(8) infected cells/bottle. The tyrosine kinase activity was retained through purification. The enzyme demonstrated much higher autophosphorylation activity in the presence of Mn2+ than Mg2+. Phosphopeptide mapping revealed the same autophosphorylation sites utilized by EGFR-IC as those identified in wild-type EGFR. EGFR-IC-catalyzed phosphorylation of either a synthetic peptide representing the major autophosphorylation site or angiotensin II showed that the baculovirus-expressed EGFR-IC exhibits similar enzymatic kinetic characteristics to the intact activated EGFR kinase.

Animals↗
PubMed1988

Biological activities of EGF-receptor mutants with individually altered autophosphorylation sites.

In vitro site-directed mutagenesis was used to replace individually the three known autophosphorylation sites of the epidermal growth factor (EGF)-receptor (i.e. Tyr1173, Tyr1148 and Tyr1068) by phenylalanine, a residue which cannot serve as a phosphate acceptor site. In another mutant, Tyr1173 was substituted by a serine residue. The cDNA constructs encoding either mutant or wild-type EGF-receptors were transfected into NIH-3T3 cells devoid of endogenous EGF-receptors. The mutant receptors were expressed on the cell surface and displayed typical high- and low-affinity binding sites for [125I]EGF. Phorbol ester (PMA) modulated the binding affinity of wild-type and mutant receptors in a similar manner. Mutant EGF-receptors exhibited EGF-dependent tyrosine kinase activity leading to self-phosphorylation and phosphorylation of exogenous substrates both in vitro and in living cells. The internalization and degradation of EGF-receptors were not affected by the mutations. Cells expressing mutant EGF-receptors became mitogenically responsive to EGF, indicating that none of the vital functions of the EGF-receptor were critically impaired by the loss of individual autophosphorylation sites. Maximal mitogenic stimulation correlated with the number of wild-type or mutant receptors per cell, highly expressing cells showing higher maximal stimulation. However, the dose-response curves of cells expressing mutant receptors were slightly shifted to lower concentrations of EGF, rendering the cells mitogenically responsive to lower doses of EGF than cells expressing normal EGF-receptor at similar expression levels. Basal [3H]thymidine incorporation in the presence of 0.5% calf serum was consistently higher for cells expressing mutant receptors, while the response to stimulation with 10% calf serum was not affected.

Animals↗
PubMed1988

Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites.

Epidermal growth factor (EGF)-receptor mutants in which individual autophosphorylation sites (Tyr1068, Tyr1148 or Tyr1173) have been replaced by phenylalanine residues were expressed in NIH-3T3 cells lacking endogenous EGF-receptors. Kinetic parameters of the kinase of wild-type and mutant receptors were compared. Both wild-type and mutant EGF-receptors had a Km(ATP) 1-3 microM for the autophosphorylation reaction, and a Km(ATP) of 3-7 microM for the phosphorylation of a peptide substrate. These are similar to the Km(ATP) values reported for EGF-receptor of A431 cells. A synthetic peptide representing the major in vitro autophosphorylation site Tyr1173 of the EGF-receptor (KGSTAENAEYLRV) was phosphorylated by wild-type receptor with a Km of 110-130 microM, and the peptide inhibited autophosphorylation with a Ki of 150 microM. Mutant EGF-receptors phosphorylated the peptide substrate with a Km of 70-100 microM. A similar decrease of Km (substrate) was obtained when the phosphorylation experiments were performed with the commonly applied substrates angiotensin II and a peptide derived from c-src. The Km of angiotensin II phosphorylation was reduced from 1100 microM for wild-type receptor to 890 microM for mutant receptor and for c-src peptide from 1010 microM to 770 microM respectively. The Vmax of the kinase was dependent on receptor concentration, but was not significantly affected by the mutation. Analogs of the Tyr1173 peptide in which the tyrosine residue was replaced by either a phenylalanine or an alanine residue also inhibited autophosphorylation with Ki of 650-750 microM. These analyses show that alterations of individual autophosphorylation sites do not have a major effect on kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1988

Fructose-1,6-bisphosphate aldolase from Drosophila melanogaster: primary structure analysis, secondary structure prediction, and comparison with vertebrate aldolases.

The amino acid sequence of fructose-1,6-bisphosphate aldolase from Drosophila melanogaster was determined and was compared with those of five vertebrate aldolases on record. The four identical polypeptide chains of the insect enzyme, acetylated at the N-terminus and three residues shorter than the vertebrate chains, contain 360 amino acid residues. Of these 190 (or 53%) are identical in all six enzymes and in addition 33 positions (or 9%) are occupied by homologous residues. Comparison with the muscle-type isoaldolases from man and rabbit and the liver-type isoaldolases from man, rat, and chicken indicates an average sequence identity of 70 and 63%, respectively. Thus, the insect and the vertebrate muscle aldolases are probably coded by orthologous genes. On this basis an average rate of evolution of 3.0 PAM per 10(8) years is calculated, documenting an evolutional divergence slower than that of cytochrome c (4.2 PAM/10(8) years). The rate is also lower than that of the liver isoform (3.6 PAM/10(8) years). Secondary structure prediction analysis for Drosophila aldolase suggests the occurrence of 11-12 helical segments and 8-9 beta-strands. The conspicuous alternation of these structures in all six aldolases, especially in the C-terminal 200 residues, is consistant with the formation of an alpha beta-barrel supersecondary structure as documented for several other glycolytic enzymes.

Amino Acid Sequence↗
PubMed1988

[A hearing implant: indications, technic and results].

The authors report four cases of implantation of a new device via an osseous approach involving implantation of an intra-osseous vibrator and of an external box, transmission being by magnetic induction using an external aerial, i.e. with no transcutaneous portion. The Xomed Audiant is an osseous rehabilitation system designed essentially for use in transmission deafness which cannot be corrected by conventional surgery or cannot be managed by an air conduction device, e.g. agenesis of the ear or sequelae of evacuation cavities in otorrhea. The authors report their experience of four cases of implantation, in terms of indications, selection assessment and evaluation protocol as well as functional results and prostatic adjustment, with a critical review of advantages and disadvantages in comparison with classical methods.

Adolescent↗
PubMed1986

Insulin-like growth factors I and II in fetal and adult bovine serum. Purification, primary structures, and immunological cross-reactivities.

Insulin-like growth factors I and II (IGF-I and IGF-II) were prepared from fetal calf serum and adult bovine serum by gel filtration, immunoaffinity chromatography, chromatofocusing, and reverse-phase high pressure liquid chromatography, and their complete amino acid sequences determined. IGF-I and -II are found in both adult and fetal serum. The sequence of bovine IGF-I is found to be identical to that of human IGF-I, whereas 3 out of 67 amino acid residues are found to be different between bovine and human IGF-II. The differences are located in the C-peptide region of the molecule. Bovine IGF-II shows less than 10% immunological cross-reactivity with antisera against human and rat IGF-II, but is equipotent to human IGF-II in displacing human 125I-labeled IGF-II from human placental receptor. Bovine IGF-I was equipotent to human IGF-I in both radioimmunoassays and radioreceptor assays within the limits of the assay.

Age Factors↗
PubMed1981

The behaviour of peptides on reverse-phase supports during high-pressure liquid chromatography.

High-pressure ('performance') liquid chromatography has been used to investigate the reverse-phase chromatographic behaviour of peptides, ranging in length from 2 to 65 amino acid residues, which have originated from primary-sequence determinations or solution/solid-phase syntheses. By using a pyridine/formate-pyridine/acetate/propan-1-ol buffer system, as previously described [Hughes, Winterhalter & Wilson (1979) FEBS Lett. 108, 81-86], the influence of various experimental parameters were examined. (a) Peptide retention was observed to be temperature-independent between 25 and 55 degrees C. (b) The dependence of chromatographic retention on pH decreases with increasing peptide hydrophobicity. (c) Chromatographic results from C8- and C18-chain-length, as well as from 5 micrometers- and 10 micrometers-particle-size, supports were comparable. (d) The hydrophobic strength of the organic solvent in the mobile phase was observed to decrease: propan-1-ol approximately equal to propan-2-ol greater than acetonitrile much greater than methanol. (e) When gradient rates (% of buffer B/unit time) were systematically decreased, peptide retention decreased in a hyperbolic manner. Comparisons of the peptides chromatographed with respect to their measured retention properties and calculated hydrophobicities were performed by computer analysis. Deviation of peptide chromatographic behaviour was observed to be essentially independent of hydrophobicity, chain length and charge. On the basis of the measured retention properties of the chromatographed peptides, hydrophobic constants for the various amino acid side chains were determined and compared with similar constants available from the literature.

Amino Acids↗
PubMed1981

Comparison of buffers and detection systems for high-pressure liquid chromatography of peptide mixtures.

The chromatographic properties of peptides ranging in length from 2 to 65 residues have been compared on reverse-phase packings in three buffer systems at low pH. Of the buffers examined, two are widely used in connection with u.v. detection [(i) phosphate/acetonitrile or (ii) phosphate/propan-2-ol] and the third for fluorescence detection [(iii) pyridine/formate-pyridine/acetate/propan-1-ol]. The addition of a chaotropic salt, NaClO4, to the phosphate buffers, as first described by Meek (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1632-1636, is shown to significantly improve the chromatographic behaviour of more hydrophobic peptides. The two most commonly used detection systems, u.v. and fluorescence, are compared in terms of ease of use and sensitivity.

1-Propanol↗