Hans Rudolf Bosshard

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.

Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition

The principles of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) are reviewed together with the basic thermodynamic formalism on which the two techniques are based. Although ITC is particularly suitable to follow the energetics of an association reaction between biomolecules, the combination of ITC and DSC provides a more comprehensive description of the thermodynamics of an associating system. The reason is that the parameters ΔG, ΔH, ΔS, and ΔCp obtained from ITC are global properties of the system under study. They may be composed to varying degrees of contributions from the binding reaction proper, from conformational changes of the component molecules during association, and from changes in molecule/solvent interactions and in the state of protonation. Copyright

Microcontact Printing of Proteins

Surface-bound mole-cules find application in biosensors, chromatography, diagnos-tic immunoassays, cell culturing, DNA microarrays, and otheranalytical procedures. Among these applications, diagnosticimmunoassays and DNA sensing are driving efforts to minia-turize biological assays and to conduct many assays in paral-lel. This trend offers several advantages. In small volumes,biochemical reactions may not be diffusion-limited and maythus be more efficient; less reagent and sample solution areused, lowering costs per test.

ANTIGEN RECOGNITION BY CONFORMATIONAL SELECTION

Conformational adaptation between antigen and antibody can modulate the antibody specificity. The phenomenon has often been proposed to result from an ‘induced fit’, which implies that the binding reaction induces a conformational change in the antigen and the antibody. Thus, an ‘induced fit’ requires initial complex formation followed by a conformational change in the complex. However, an antibody may select those antigen molecules that happen to be in a fitting conformational state. This leads to the same end result as an induced fit. Here, we demonstrate conformational selection by a single chain antibody fragment, raised against a random coil variant of the leucine zipper domain of transcription factor GCN4, when it cross‐reacts with the wild‐type dimeric leucine zipper. Kinetic and equilibrium data show that the single chain antibody fragment fragment selects monomeric peptides from the population in equilibrium with the leucine zipper dimer.

Fabricating Microarrays of Functional Proteins Using Affinity Contact Printing

Phenomena involving the binding between biomolecules are ubiquitous in biology and are essential for cell growth, signal transmission, and immune defense. In the latter system, the binding between antibody and antigen has already been exploited technologically to perform affinity purifications on columns and immunoassays on surfaces.[1] Recently, the fabrication of microarrays of proteins which require the immobilization of a large number of receptors on a surface have fueled the invention of novel patterning techniques such as pin-spotting and drop-on-demand.[2] Microarrays of proteins may find utility in proteomics, immunoassays, or for screening libraries of (bio)chemicals. It is at present not clear which patterning method will be the one best suited to pattern proteins on surfaces, but classical lithography does not seem capable of fabricating microarrays of proteins. Soft lithography[3] offers the possibility of manipulating proteins and other biomolecules by printing them from a micropatterned stamp to a surface[4] or by depositing them from a liquid using microfluidic networks ( FNs).[5] Affinity microcontact printing ( CP)[6] is a refined soft-lithographic technique that uses an elastomeric stamp made of polydimethylsiloxane (PDMS) and derivatized with binding biomolecules to extract corresponding binding partners from an impure, dilute source for placing them on a surface with spatial control. Herein, we describe, by using one particular example, how specific binding between biomolecules provides a unique opportunity to make use of self-assembly processes in technology: we propose different variants of CP to pattern surfaces with ensembles of biomolecules where the pattern on the affinity stamp ( -stamp) is not determined by its topography but by the position of various proteins covalently linked to a planar -stamp (Figure 1). This modified surface enables the simultaneous capture of different target proteins on the -stamp from a complex solution (Figure 1A). Thus, the capture step (Figure 1B) directs the assembly of an array of target molecules on the stamp (Figure 1C), which can be Figure 1. Microarrays of proteins on surfaces can be fabricated using an stamp derivatized with various capture sites that can extract target biomolecules from a complex solution and release them on a surface in a single microcontact-printing step. The -stamp can be reused for several inking and printing cycles.

Affinity capture of proteins from solution and their dissociation by contact printing

Biological experiments at the solid/liquid interface, in general, require surfaces with a thin layer of purified molecules, which often represent precious material. Here, we have devised a method to extract proteins with high selectivity from crude biological sample solutions and place them on a surface in a functional, arbitrary pattern. This method, called affinity-contact printing (αCP), uses a structured elastomer derivatized with ligands against the target molecules. After the target molecules have been captured, they are printed from the elastomer onto a variety of surfaces. The ligand remains on the stamp for reuse. In contrast with conventional affinity chromatography, here dissociation and release of captured molecules to the substrate are achieved mechanically. We demonstrate this technique by extracting the cell adhesion molecule neuron-glia cell adhesion molecule (NgCAM) from tissue homogenates and cell culture lysates and patterning affinity-purified NgCAM on polystyrene to stimulate the attachment of neuronal cells and guide axon outgrowth.

Caveats for the use of surface-adsorbed protein antigen to test the specificity of antibodies

Rabbit antisera against apo-cytochrome c, which was prepared by removal of the covalently bound heme prosthetic group from yeast iso-1 cytochrome c, were tested for reactivity against native yeast iso-1-cytochrome c. When the antigen was adsorbed to a microtiter plate in a conventional enzyme-linked immunosorbent assay (ELISA), the antisera were unable to distinguish between their cognate antigen apo-cytochrome c, a random coil protein, and native cytochrome c, a small globular protein of remarkable conformational stability in solution. However, when the assay was conducted under conditions where antigen and antibody were free to associate in solution, that is in a solution-phase radioimmunoassay (RIA), the antisera were highly specific for apo-cytochrome c. Similarly, antibodies induced by native cytochrome c and discriminating strongly between native and apo-cytochrome c in a solution-phase RIA, did not distinguish between native and apo-cytochrome c in a solid-phase ELISA. This discrepancy of results obtained by different immuno assay procedures clearly indicates that adsorption to plastic alters the antigenic structure of even a conformationally stable protein such as cytochrome c. A conventional solid-phase ELISA strongly selects for those antibodies that recognize the unfolded antigen. The results presented warrant serious thoughts about previous reports on anti-peptide antibodies reacting with native whole protein molecules, as tested by those ELISA procedures that have the protein antigen adsorbed to plastic.

Protein A antibody-capture ELISA (PACE): an ELISA format to avoid denaturation of surface-adsorbed antigens

Adsorption to a polymeric surface may severely alter the antigenic structure of proteins through unfolding. A conventional capture ELISA in which a protein antigen is adsorbed to the microtiter plate may be unsuitable for testing the specificity of antibodies directed against native proteins (C. Schwab and H.R. Bosshard (1992) J. Immunol. Methods 147, 125). This problem can be overcome by PACE, a new ELISA procedure in which monoclonal or polyclonal antibodies are first allowed to equilibrate with biotinylated antigen in solution. Thereafter, the antigen-antibody complex (and free antibody) is bound to the microtiter plate through protein A. Captured antigen-antibody complex is detected by streptavidin-alkaline phosphatase and p-nitrophenylphosphate. A competition assay is accomplished by co-incubation of biotinylated and non-biotinylated antigens before capture to the protein A-coated plate. PACE combines the advantages of a solution-phase immunoassay (Farr assay) with the ease of a solid-phase ELISA. PACE has been used to test the conformational specificity of polyclonal and monoclonal antibodies against native and denatured cytochrome c, and of a polyclonal antiserum against a coiled coil leucine zipper peptide. Since a biotin group can be attached specifically to the N-terminal residue of synthetic peptides, PACE is also useful for assaying reactivity against peptide antigens which are difficult to adsorb to microtiter plates.

Calculation of protein ionization equilibria with conformational sampling: pKa of a model leucine zipper, GCN4 and barnase †

The use of conformational ensembles provided by nuclear magnetic resonance (NMR) experiments or generated by molecular dynamics (MD) simulations has been regarded as a useful approach to account for protein motions in the context of pKa calculations, yet the idea has been tested occasionally. This is the first report of systematic comparison of pKa estimates computed from long multiple MD simulations and NMR ensembles. As model systems, a synthetic leucine zipper, the naturally occurring coiled coil GCN4, and barnase were used. A variety of conformational averaging and titration curve‐averaging techniques, or combination thereof, was adopted and/or modified to investigate the effect of extensive global conformational sampling on the accuracy of pKa calculations. Clustering of coordinates is proposed as an approach to reduce the vast diversity of MD ensembles to a few structures representative of the average electrostatic properties of the system in solution. Remarkable improvement of the accuracy of pKa predictions was achieved by the use of multiple MD simulations. By using multiple trajectories the absolute error in pKa predictions for the model leucine zipper was reduced to as low as approximately 0.25 pKa units. The validity, advantages, and limitations of explicit conformational sampling by MD, compared with the use of an average structure and a high internal protein dielectric value as means to improve the accuracy of pKa calculations, are discussed. Proteins 2002;46:41–60.

Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges.

Electrostatic interactions play a complex role in stabilizing proteins. Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper AB(SS). The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pK(a) induced by folding of the coiled coil structure. pK(a)(F) values of ten Glu and two His side-chains in folded AB(SS) and the corresponding pK(a)(U) values in unfolded peptides with partial sequences of AB(SS) were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (-5.6 kJ mol(-1)) and two are interacting with the positive alpha-helix dipoles and are thus stabilizing (+3.8 kJ mol(-1)) in charged form. The two His residues positioned in the C-terminal moiety of AB(SS) interact with the negative alpha-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (-2.6 kJ mol(-1)). Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (-1.1 kJ mol(-1)). The sum of the individual contributions of protonated Glu and His to the higher stability of AB(SS) at acidic pH (-5.4 kJ mol(-1)) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (-5.3 kJ mol(-1)). To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys-->norleucine, Arg-->norvaline). Both mutations destabilize the coiled coil conformation at neutral pH and increase the pK(a) of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing. Because removing charges by a double mutation cycle mainly discloses the immediate charge-charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability.