PubMed Health⌕ Search

Biomedical subjects

A Skerra

Publications and source records attributed to A Skerra.

At least 55 records · Page 3Linked to original sources
PubMed1994

A general vector, pASK84, for cloning, bacterial production, and single-step purification of antibody Fab fragments.

The expression vector pASK84 was designed for the convenient cloning of immunoglobulin variable domain genes, as well as periplasmic secretion of the corresponding F(ab) fragment in Escherichia coli. The plasmid provides the constant domain genes of mouse IgG1/kappa with a hexa-histidine tag fused to the C terminus of the heavy chain. This strategy enables the rapid and efficient purification of the functional recombinant F(ab) fragment via immobilized metal affinity chromatography. The versatility of this expression and purification system is demonstrated using the variable domains of the well-characterized anti-lysozyme antibody D1.3.

Amino Acid Sequence↗
PubMed1994

The bilin-binding protein of Pieris brassicae. cDNA sequence and regulation of expression reveal distinct features of this insect pigment protein.

The bilin-binding protein (BBP) is a blue pigment protein which is abundant in the butterfly Pieris brassicae. In an attempt to clarify the physiological role of this member of the lipocalin family of proteins, its complete cDNA was cloned and expression of the BBP gene in P. brassicae was investigated. It was found that synthesis of the BBP mRNA is highly regulated during the insect's ontogenesis. In larvae after the third ecdysis as well as in pupae and adults, large amounts of the mRNA are present. Each of these stages itself displays a distinct time course of mRNA synthesis. In addition, BBP is expressed tissue specifically, with the fat body being the major source of this secretory protein in the larvae. Hence, the expression pattern of BBP in this organism is markedly different from the closely related pigment protein insecticyanin in Manduca sexta. Finally, the bacterial expression of BBP in a functional state was established as a basis for the future analysis of its ligand-binding functions by protein engineering.

Amino Acid Sequence↗
PubMed1993

Single-step purification of a bacterially expressed antibody Fv fragment by immobilized metal affinity chromatography in the presence of betaine.

A procedure was developed for the rapid isolation of an antibody Fv fragment expressed in Escherichia coli via immobilized metal affinity chromatography. Metal affinity was mediated by fusing hexahistidine tails to both the VL and the VH domain and was thus independent of the antigen-binding specificity. Unexpectedly, it was not possible to isolate the Fv fragment with correct stoichiometric composition of the two variable domains under standard chromatographic conditions. Proper non-covalent association of VL and VH was, however, maintained when using glycine betaine as electrolyte, thus permitting purification of the intact Fv fragment to homogeneity in a single step.

Antibody Specificity↗
PubMed1993

Functional expression of the uncomplexed serum retinol-binding protein in Escherichia coli. Ligand binding and reversible unfolding characteristics.

The serum retinol-binding protein solubilizes the lipophilic vitamin A alcohol and plays an important physiological role in the transport of this compound. The monomeric single-domain protein, the three-dimensional structure of which is known, constitutes a well-characterized member of the lipocalin family of proteins. We report here the functional expression of the apo-protein in Escherichia coli by secretion to the periplasm. The recombinant protein, purified in a single step by metal chelate affinity chromatography, exhibits the same ligand binding characteristics as described for the natural protein. Guanidinium chloride-induced unfolding and refolding experiments suggest that the recombinant retinol-binding protein adopts a stable conformation despite being expressed and purified in the absence of the large hydrophobic ligand. The expression system described here should also be useful for the recombinant production of other lipocalin proteins, thus permitting the elucidation of the structure-function relationships of ligand binding by protein engineering.

Amino Acid Sequence↗
PubMed1993

Bacterial expression of immunoglobulin fragments.

The expression of Ig fragments in Escherichia coli permits rapid access to engineered molecules with antigen-binding properties. While the expression in a functional state by secretion to the periplasm is the standard method for the production of Fv and Fab fragments, single chain Fv fragments are mainly produced by refolding from insoluble aggregates. Although all of these Ig fragments serve as valuable aids in the study of antigen binding, their different biochemical properties must be considered when using them as research tools or for medical applications. In addition to these simple univalent antibody fragments, the bacterial expression of bivalent and bispecific versions and of hybrid proteins with novel effector functions is gaining increasing importance.

Animals↗
PubMed1993

The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment.

The facile detection and purification of a recombinant protein without detailed knowledge about its individual biochemical properties constitutes a problem of general interest in protein engineering. The use of a novel kind of random peptide library for the stepwise engineering of a C-terminal fusion peptide which confers binding activity towards streptavidin is described in this study. Because of its widespread use as part of a variety of conjugates and other affinity reagents, streptavidin constitutes the binding partner of choice both for detection and purification purposes. The streptavidin-affinity tag was engineered at the C-terminus of the VH domain as part of the D1.3 Fv fragment which was functionally expressed in Escherichia coli. Irrespective of whether it was displayed by the VH or the VL domain, the optimized version of the affinity peptide termed 'Strep-tag' allowed the detection of the Fv fragment both on Western blots and in ELISAs by a streptavidin-alkaline phosphatase conjugate. In addition, the one-step purification of the intact Fv fragment carrying a single Strep-tag at the C-terminus of only one of its domains was achieved by affinity chromatography with streptavidin-agarose using very mild elution conditions.

Affinity Labels↗
PubMed1992

Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity.

Two thermostable DNA polymerases with proofreading activity--Vent DNA polymerase and Pfu DNA polymerase--have attracted recent attention, mainly because of their enhanced fidelities during amplification of DNA sequences by the polymerase chain reaction. A severe disadvantage for their practical application, however, results from the observation that due to their 3' to 5' exonuclease activities these enzymes degrade the oligodeoxynucleotides serving as primers for the DNA synthesis. It is demonstrated that this exonucleolytic attack on the primer molecules can be efficiently prevented by the introduction of single phosphorothioate bonds at their 3' termini. This strategy, which can be easily accomplished using routine DNA synthesis methodology, may open the way to a widespread use of these novel enzymes in the polymerase chain reaction.

Amino Acid Sequence↗
PubMed1991

Colony assays for antibody fragments expressed in bacteria.

This paper describes procedures for the detection and selection of bacterial colonies expressing antibody fragments of desired antigen specificity. Fab and Fv fragments are detected in a filter assay in which bacterial colonies are grown on a master filter in contract with a second, antigen-coated filter. Ab fragments diffusing onto the second filter bind antigen directly and specifically and are detected with a monoclonal antibody directed against a myc-tag sequence fused to the carboxy-terminal end of the light chain or heavy chain (direct assay). Single-chain Fv (scFv) in which the VH and V1 sequences are joined by a short linker peptide are detected by a modified procedure in which scFv are immobilized on filters coated with the anti-myc-tag sequence and subsequently detected by specific binding to radiolabeled antigen (indirect assay). A single positive bacterial colony expressing antigen-specific Fv (or scFv) can be recovered among at least 10,000 negative colonies using the procedures described. The direct assay has been successfully used to discriminate Fv fragments which express point mutations known to increase the binding affinity of antibodies to the hapten 2-phenyl-oxazolone. The procedures described may thus prove generally useful for the selection of antigen-specific clones expressed in bacteria and/or higher-affinity variants of such antibodies.

Antibodies↗
PubMed1991

Filter screening of antibody Fab fragments secreted from individual bacterial colonies: specific detection of antigen binding with a two-membrane system.

Recently antibody fragments have been expressed in a functional form from bacteria. We have devised a simple method to detect the binding of antigen to antibody Fab fragments secreted by bacterial colonies. Bacteria harboring plasmid vectors that direct the secretion of Fab fragments into the bacterial periplasm are grown on one membrane. The secreted fragments are allowed to diffuse to a second "capture" membrane coated with anti-globulin, and are probed with antigen. Using enzyme or colloidal gold conjugates, the binding of antigen is detected on the second membrane as a colored spot. The colonies can be regrown on the first membrane, and the antigen binding signal on the second membrane is free of noise contributed by bacterial debris.

Antigen-Antibody Reactions↗
PubMed1991

The functional expression of antibody Fv fragments in Escherichia coli: improved vectors and a generally applicable purification technique.

We have previously demonstrated that the expression of fully functional Fv and Fab fragments in E. coli is possible by the simultaneous secretion of both chains to the periplasm. To increase production levels and facilitate engineering and random mutagenesis, we improved our previous vectors by introducing a resident repressor gene and a filamentous phage origin. We also developed a new purification strategy based on immobilized metal ion chromatography, with which a single-chain Fv fragment can be purified to homogeneity in a single step. We investigated the most efficient tail constructions and found that only a minimal structural change of three additional C-terminal amino acids is necessary. This modification has no deleterious effect on in vivo transport and folding or antigen affinity.

Amino Acid Sequence↗
PubMed1991

Secretion and in vivo folding of the Fab fragment of the antibody McPC603 in Escherichia coli: influence of disulphides and cis-prolines.

Using the well-characterized antibody McPC603 as a model, we had found that the Fv fragment can be isolated from Escherichia coli as a functional protein in good yields, whereas the amount of the correctly folded Fab fragment of the same antibody produced under identical conditions is significantly lower. In this paper, we analyse the reasons for this difference. We found that a variety of signal sequences function in the secretion of the isolated chains of the Fab fragment or in the co-secretion of both chains in E.coli. The low yield of functional Fab fragment is not caused by inefficient expression or secretion in E.coli, but by inefficient folding and/or assembly in the periplasm. We compared the folding yields for the Fv and the Fab fragment in the periplasm under various conditions. Several diagnostic framework variants were constructed and their folding yields measured. The results show that substitutions affecting cis-proline residues and those affecting various disulphide bonds in the protein are by themselves insufficient to dramatically change the partitioning of the folding pathway to the native structure, and the cause must lie in a facile aggregation of folding intermediates common to all structural variants. However, all structural variants could be obtained in native form, demonstrating the general utility of the secretory expression strategy.

Amino Acid Sequence↗
PubMed1990

Structural features of the McPC603 Fab fragment not defined in the X-ray structure.

The proteolytic Fab fragment of the well characterized antibody McPC603 was compared to the recombinant Fab fragment, which was obtained in functional form from an Escherichia coli expression system [(1989) Methods Enzymol. 178, 497-515]. We found evidence that the proteolytic fragment is glycosylated at Asn H160 in the CH1 domain, where additional electron density had been observed in the crystal structure [J. Mol. Biol. 190, 593-604]. In addition, its heavy chain is about 30 amino acids longer than visible in the electron density and thus contains the complete hinge region. These structural differences between the recombinant Fab fragment, which had been designed exactly according to the defined electron density, and the proteolytic Fab fragment of McPC603 had no effect on the hapten binding properties of these antigen binding fragments. Yet, it may be important to be aware of these structural features of McPC603 in folding studies and some comparative analyses of antibody structures.

Amino Acid Sequence↗
PubMed1990

Properties of FV and Fab fragments of the antibody McPC603 expressed in E. coli.

The FV and Fab fragments of the phosphorylcholine binding antibody McPC603 were functionally expressed in E. coli. This was achieved by the co-expression and co-secretion of both chains to the periplasm, where correct processing, folding and assembly occurred. Interestingly, the fraction of correctly folded Fab fragment is smaller than that of the Fv fragment in E. coli. The intrinsic hapten binding affinity was shown to be identical for the recombinant FV or Fab fragment, the whole antibody and the Fab fragment obtained by proteolysis from the mouse antibody. Fluorescence and crosslinking analyses showed that the FV fragment dissociates at high dilution, but that it is stabilized by hapten binding. The recombinant FV fragment was shown to have catalytic activity to hydrolyze choline-p-nitrophenyl carbonate and constitutes therefore a promising model system with which the structural requirements of catalytic antibodies can be studied by altering the protein itself.

Animals↗
PubMed1988

Assembly of a functional immunoglobulin Fv fragment in Escherichia coli.

An expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in Escherichia coli. The variable domains of the phosphorylcholine-binding antibody McPC603 were secreted together into the periplasmic space, where protein folding as well as heterodimer association occurred correctly. Thus, the assembly pathway for the Fv fragment in E. coli is similar to that of a whole antibody in the eukaryotic cell. The Fv fragment of McPC603 was purified to homogeneity with an antigen-affinity column in a single step. The correct processing of both signal sequences was confirmed by amino-terminal protein sequencing. The functionality of the recombinant Fv fragment was demonstrated by equilibrium dialysis. These experiments showed that the affinity constant of the Fv fragment is identical to that of the native antibody McPC603, that there is one binding site for phosphorylcholine in the Fv fragment, and that there is no inactive protein in the preparation. This expression system should facilitate future protein engineering experiments on antibodies.

Escherichia coli↗
PubMed1987

Structure and dynamics of one-dimensional ionic solutions in biological transmembrane channels.

The structure and dynamics of solvated alkali metal cations in transmembrane channels are treated using the molecular dynamics simulation technique. The simulations are based on a modified Fischer-Brickmann model (Fischer, W., and J. Brickmann, 1983, Biophys. Chem., 18:323-337) for gramicidin A-type channels. The trajectories of all particles in the channel as well as two-dimensional pair correlation functions are analyzed. It is found from the analysis of the stationary simulation state that one-dimensional solvation complexes are formed and that the number of water molecules in the channel varies for different alkali metal cations.

Ion Channels↗
PubMed1987

Simulation of voltage-driven hydrated cation transport through narrow transmembrane channels.

Molecular dynamics studies for the voltage-driven transport of the alkali metal ions lithium, sodium, and potassium through gramicidin A-type channels filled with water molecules are presented. The number of water molecules in the channel is obtained from a previous study (Skerra, A., and J. Brickmann, 1987, Biophys. J., 51:969-976). It is shown that the selectivity of the intrachannel ion diffusion through our model pore conforms to the experimentally observed selectivity of the gramicidin A channel. It is demonstrated that the number of water molecules in the channel plays a key role for the selectivity.

Cations↗