PubMed Health⌕ Search

Biomedical subjects

K Nord

Publications and source records attributed to K Nord.

11 recordsLinked to original sources
PubMed2001

Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A.

Factor VIII-specific affibodies were selected from phage displayed libraries constructed by combinatorial mutagenesis of an alpha helical bacterial receptor domain derived from staphylococcal protein A. Bead-immobilized recombinant human factor VIII (rVIII) (80 and 90 kDa chains) protein was used during competitive biopannings in the presence of free 80-kDa chain protein, resulting in the selection of several binders that showed dissociation constants (Kd) in the range 100-200 nM as determined by biosensor analyses. One variant (Z[rVIII:3], 90-kDa chain specific) was further characterized in small-scale affinity chromatography experiments, and showed efficient and selective recovery of biologically active rVIII from Chinese hamster ovary cell supernatant-derived feed stocks. The purity of the enriched rVIII was comparable with rVIII material purified by immunoaffinity chromatography using a 90-kDa chain-specific monoclonal antibody. Interestingly, epitope mapping showed that the monoclonal antibody and the affibody ligand competed for the same or at least overlapping epitopes on rVIII. In addition, the Z[rVIII:3] variant was produced by peptide synthesis with a C-terminal cysteine to enable directed coupling to solid supports. This 59-residue protein was analyzed by circular dichroism and showed a secondary structure content similar to that of the parental Z domain used as scaffold. In biosensor studies, the synthetic affibody was immobilized recruiting the C-terminal cysteine residue, and demonstrated to bind both recombinantly produced and plasma-derived factor VIII. From a secondary library, constructed by re-randomization of relevant positions identified after alignment of the first-generation variants, a panel of affinity-improved second-generation affibodies were selected of which one clone showed a dissociation constant (Kd) for rVIII of 5 nM. Several of these variants also showed higher apparent binding efficiencies towards rVIII when analyzed as immobilized ligands in biosensor experiments. Taken together, the results suggest that affibody ligands produced by bacterial or synthetic routes could be of interest as an alternative to monoclonal antibodies in purification processes or as diagnostic or monitoring tools.

Amino Acid Sequence↗
PubMed2000

Ligands selected from combinatorial libraries of protein A for use in affinity capture of apolipoprotein A-1M and taq DNA polymerase.

Here we show that robust and small protein ligands can be used for affinity capture of recombinant proteins from crude cell lysates. Two ligands selectively binding to bacterial Taq DNA polymerase and human apolipoprotein A-1(M), respectively, were used in the study. The ligands were selected from libraries of a randomized alpha-helical bacterial receptor domain derived from staphylococcal protein A and have dissociation constants in the micromolar range, which is typical after primary selection from these libraries consisting of approximately 40 million different members each. Using these ligands in affinity chromatography, both target proteins were efficiently recovered from crude cell lysates with high selectivities. No loss of column capacity or selectivity was observed for repeated cycles of sample loading, washing and low pH elution. Interestingly, column sanitation could be performed using 0. 5 M sodium hydroxide without significant loss of ligand performance. The results suggest that combinatorial approaches using robust protein domains as scaffolds can be a general tool in the process of designing purification strategies for biomolecules.

Amino Acid Sequence↗
PubMed1999

Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling.

The possibility of increasing the affinity of a Taq DNA polymerase specific binding protein (affibody) was investigated by an alpha-helix shuffling strategy. The primary affibody was from a naive combinatorial library of the three-helix bundle Z domain derived from staphylococcal protein A. A hierarchical library was constructed through selective re-randomization of six amino acid positions in one of the two alpha-helices of the domain, making up the Taq DNA polymerase binding surface. After selections using monovalent phage display technology, second generation variants were identified having affinities (K(D)) for Taq DNA polymerase in the range of 30-50 nM as determined by biosensor technology. Analysis of binding data indicated that the increases in affinity were predominantly due to decreased dissociation rate kinetics. Interestingly, the affinities observed for the second generation Taq DNA polymerase specific affibodies are of similar strength as the affinity between the original protein A domain and the Fc domain of human immunoglobulin G. Further, the possibilities of increasing the apparent affinity through multimerization of affibodies was demonstrated for a dimeric version of one of the second generation affibodies, constructed by head-to-tail gene fusion. As compared with its monomeric counterpart, the binding to sensor chip immobilized Taq DNA polymerase was characterized by a threefold higher apparent affinity, due to slower off-rate kinetics. The results show that the binding specificity of the protein A domain can be re-directed to an entirely different target, without loss of binding strength.

Antibodies↗
PubMed1998

Photoreactivity of biologically active compounds, XIV: influence of oxygen on light induced reactions of primaquine.

The influence of molecular oxygen and oxygen radicals on the photoreactivity of the antimalarial drug primaquine (PQ) has been investigated. Oxygen is directly involved in photodecomposition of the drug. Flushing with helium gas prior to and during irradiation to suppress the oxygen level of the medium, retards the degradation rate of PQ (followed by HPLC) and leads to the formation of only two degradation products (identified by MS) compared to eight main- and several minor products under normal atmospheric conditions. Flushing with oxygen gas prior to and during irradiation to increase the oxygen content of the medium accelerates the degradation rate of PQ. PQ produces oxygen radicals (hydroxyl and superoxide) during photolysis, while the photoproducts of PQ seem likely to induce singlet oxygen formation (detected by addition of radical scavengers). Sensitization reactions involving singlet oxygen lead to decomposition of PQ (followed by HPLC). On the basis of our results, photochemical reaction mechanisms of PQ are postulated and discussed. At physiological conditions (aqueous, neutral pH, oxygen rich) PQ has a large potential to decompose after light absorption. The photoreaction seems to be initiated at the quinoline nitrogen. The ability to form an intramolecular hydrogen bond seems to be essential for the luminescence properties of the drug. Phosphorescence lifetime of PQ is about 5 microseconds. Fast chemical reactions may occur from the short-lived triplet state of the drug, but the excited compound can diffuse only a limited distance prior to deexcitation. This can be important concerning light-induced adverse effects which may appear after medication with PQ.

Antimalarials↗
PubMed1998

Control of caprine arthritis-encephalitis virus and Corynebacterium pseudotuberculosis infection in a Norwegian goat herd.

A control programme for caprine arthritis-encephalitis virus (CAEV) and Corynebacterium pseudotuberculosis (C. pseudotuberculosis) infection was established in a Norwegian goat herd comprising approximately 100 milking goats. The herd seroprevalences of antibodies against CAEV and C. pseudotuberculosis were 97% and 94%, respectively. Kids were removed from the infected flock at birth, avoiding any contact between dam and kid. The kids were kept completely segregated from the seropositive flock and fed cow's colostrum and milk. A seronegative flock was established, based on the removed kids and their offspring. Goasts belonging to the seronegative flock were allowed to kid naturally and to mother their kids. The seropositive flock was slaughtered during the second year of the control programme. After washing and disinfection, housing systems and nearby outdoor premises were left empty for 3 months. Of 230 goats examined for antibodies against CAEV with ELISA regularly during 3 years of the control program, altogether 6 were found to be seropositive, while for 10 the result was indeterminate. All 16 animals were immediately culled. During the third year of the control programme, all goats were examined and proved negative for antibodies against C. pseudotuberculosis by a haemolysis inhibition test. Clinical examination revealed no signs of CAE or caseous lymphadenitis.

Animal Husbandry↗
PubMed1997

Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain.

Small protein domains, capable of specific binding to different target proteins have been selected using combinatorial approaches. These binding proteins, called affibodies, were designed by randomization of 13 solvent-accessible surface residues of a stable alpha-helical bacterial receptor domain Z, derived from staphylococcal protein A. Repertoires of mutant Z domain genes were assembled and inserted into a phagemid vector adapted for monovalent phage display. Two libraries, each comprising approximately 4 x 10(7) transformants, were constructed using either an NN(G/T) or an alternative (C/A/G)NN degeneracy. Biopanning against the target proteins Taq DNA polymerase, human insulin, and a human apolipoprotein A-1 variant, showed that in all cases significant enrichments were obtained by the selection procedures. Selected clones were subsequently expressed in Escherichia coli and analyzed by SDS-PAGE, circular dichroism spectroscopy, and binding studies to their respective targets by biospecific interaction analysis. The affibodies have a secondary structure similar to the native Z domain and have micromolar dissociation constants (KD) for their respective targets.

Amino Acid Sequence↗
PubMed1997

Effects of infection by caprine arthritis-encephalitis virus on milk production of goats.

The effects of caprine arthritis-encephalitis virus on lactational performance of goats were examined. The results of an ELISA for antibodies against caprine arthritis-encephalitis virus were compared with milk production records. Mean production of milk, protein, fat, and lactose and somatic cell counts were compared for seropositive and seronegative goats of similar ages. The results from 1799 lactating goats from 66 herds suggested that milk production was similar for 1-yr-old goats that tested seropositive and those that tested seronegative. For 900 of those goats for which data permitted comparison, milk fat and protein were also similar. A comparison of 331 goats showed that lactose contents did not differ between 1- and 2-yr-old goats, but somatic cell counts were higher in 2-yr-old seropositive goats.

Animals↗
PubMed1995

A combinatorial library of an alpha-helical bacterial receptor domain.

The construction and characterization of a combinatorial library of a solvent-exposed surface of an alpha-helical domain derived from a bacterial receptor is described. Using a novel solid-phase approach, the library was assembled in a directed and successive manner utilizing single-stranded oligonucleotides containing multiple random substitutions for the variegated segments of the gene fragment. The simultaneous substitution of 13 residues to all 20 possible amino acids was carried out in a region spanning 81 nucleotides. The randomization was made in codons for amino acids that were modelled to be solvent accessible at a surface made up from two of the three alpha-helices of a monovalent Fc-binding domain of staphylococcal protein A. After cloning of the PCR-amplified library into a phagemid vector adapted for phage display of the mutants, DNA sequencing analysis suggested a random distribution of codons in the mutagenized positions. Four members of the library with multiple substitutions were produced in Escherichia coli as fusions to an albumin-binding affinity tag derived from streptococcal protein G. The fusion proteins were purified by human serum albumin affinity chromatography and subsequently characterized by SDS-electrophoresis, CD spectroscopy and biosensor analysis. The analyses showed that the mutant protein A derivatives could all be secreted as soluble full-length proteins. Furthermore, the CD analysis showed that all mutants, except one with a proline introduced into helix 2, have secondary structures in close agreement with the wild-type domain. These results proved that members of this alpha-helical receptor library with multiple substitutions in the solvent-exposed surface remain stable and soluble in E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)

Adhesins, Bacterial↗