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Bacterial expression of a human Fv fragment IgM with a novel antibody combining site
280
Abstracts
structures. Spatial restraints on the target sequence are obtained from the statistical analysis of the relationships between various features of protein structure. A database of 105 family alignments including 416 proteins with known 3D structure was constructed to obtain the tables quantifying the relationships, such as those between two equivalent Co - - Co distances, or between equivalent main chain dihedral angles from two related proteins. These relationships were expressed as conditional probability density distributions and can be used directly as spatial restraints. For example, probabilities for different values of the main chain dihedral angles are calculated from the type of a residue considered, from main conformation of an equivalent residue, and from sequence similarity between the two proteins. Next, the homology-derived restraints and energy terms enforcing proper stereochemistry are combined into an objective function. Finally, the model is obtained by optimizing the objective function in Cartesian space. This optimization is carried out by the use of the variable target function method, employing methods of conjugate gradients and molecular dynamics with simulated annealing. Several slightly different models can be calculated by varying the initial structure. [1] ~ali, A. and Blundell, T.L. (1993) Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779-815. [2] gali, A. and Overington, J.P. (1994) Derivation of rules for comparative protein modeling from a database of protein structure alignments. Prot. Sci. 3, 1582-1596. [3] gali, A. (1995) Modelling mutations and homologous proteins. Curr. Opin. Biotech 6, 437-451. [4] gala, A., Potterton, L., Yuan, F., van Vlijmen, H. and Karplus, M. Evaluation of comparative protein modelling by MODELLER. Protein, in press. Residues near the amino termini contribute to hapten recognition in a cloned anti-17p-estradiol Fab fragment.
Petri Saviranta, Piitu Jauria, Maria Pajunen, Urpo Lamminmfiki, Timo LSvgen, Department of Biotechnology, University
of Turku, FIN-20520 Turku, Finland. We have cloned and sequenced the Fd and kappa chains of a monoclonal antibody against 17fl-estradiol is order to study the binding site at the molecular level. The chains were cloned into the vector pComb3 [1] which was used both for Fab production and for phage display in E. coll. The cloning sites in the vector are designed for the in-frame fusion of the amino termini of the mature Fd and light chains to pelB signal sequences, to enable secretion to periplasm where Fab assembly occurs. The integrity of the recombinant Fab was assessed by comparison of its binding characteristics to a proteolytic Fab fragment of the parental hybridoma IgG. The affinity for estradiol was 2-fold decreased and cross-reactivity to testosterone was 3.5-fold increased. Since the cloning sites for the Fd and the light chain in the pComb3 vector were embedded in the framework 1 coding sequence, the amino termini of the chains were not strictly authentic. Keeping this as a tentative explanation for the observed changes in steroid binding, we
removed the embedded cloning sites to restore the original coding sequences. The revised Fab showed binding properties identical with the proteolytic fragment, confirming that the observed differences resulted from changes in the amino termini. Furthermore, restoration of the heavy chain alone affected for estradiol while the restoration of the heavy chain alone affected only testosterone cross-reactivity. The restoration of the N-Terminal sequences also improved the Fab yield and immunoreactivity by a factor of 3, primarily attributed to the influence of the heavy chain N-terminus. We have shown that the very first N-terminal residues of the antibody are able to modify hapten binding, despite the fact that they cannot be in direct contact with the cleft-bound hapten. [1] Barbas et al. (1991) Proc. Nat. Acad. Sci. USA 88, 797882) Bacterial expression of a human Fv fragment IgM with a novel antibody combining site.
Grant I. Shoebridge, Robert L. Raison, Department of Cell and Molecular Biology, Immunobiology Unit, University of Technology Sydney, Australia. The third complementarity determining region of the heavy chain (HCDR3) is a major contributor to the architecture of the antibody combining site and hence the antigen binding specificity of an immunoglobulin molecule. We have previously described an unusual HCDR3 structure from a human IgM paraprotein Pot, obtained from a patient with Waldenstrom's macroglobulinemia [1]. Pot exhibits ployreactive properties binding to several structurally unrelated antigens. Crystallographic data indicates the Pot, Fv does not have a binding site shaped like a pocket or a groove. The HCDR3 fills the space between the VH/VL interface, blocking any binding site cavity. POT Fv may therefore serve as a model system for studying thr correlation between the HCDR3 primary amino acid sequence, three dimensional structure and polyreactive binding properties. In order to express Pot Fv, a dicistronic expression vector has been constructed. Secretion signals Omp A and Pel B direct export of the VH and VL into the bacterial periplasm respectively. Optimisation of expression conditions produced yields of Img/L isolated from the culture supernatant CDR grafting experiments and site directed mutagenesis of the HCDR3 will allow identification of the major structural correlates the polyrcactivity. Comparison of the structural and binding properties of the mutagenised POT Fv with native Pot Fv will enhance our understanding of the topology of the antibody combining site, particularly with respect to polyreactivity [1] Fan, Z-C et al. (1992) J. Mol. Biol. 228, 188. Effects of mutations in the heavy chain CDR3 region on the affinity of an anti-PSA diabody.
Liming Shu, Roger Smith, Ralph Abraham, John Link, Colleen Venti, Michael Darsley, JGEN, Inc. 16020 Industrial
Drive, Gaithersburg, MD 20877, USA. Prostate specific antigen (PSA) is a 33 KD glycoprotein of a single peptide chain. It is produced exclusively by prostate
Abstracts
structures. Spatial restraints on the target sequence are obtained from the statistical analysis of the relationships between various features of protein structure. A database of 105 family alignments including 416 proteins with known 3D structure was constructed to obtain the tables quantifying the relationships, such as those between two equivalent Co - - Co distances, or between equivalent main chain dihedral angles from two related proteins. These relationships were expressed as conditional probability density distributions and can be used directly as spatial restraints. For example, probabilities for different values of the main chain dihedral angles are calculated from the type of a residue considered, from main conformation of an equivalent residue, and from sequence similarity between the two proteins. Next, the homology-derived restraints and energy terms enforcing proper stereochemistry are combined into an objective function. Finally, the model is obtained by optimizing the objective function in Cartesian space. This optimization is carried out by the use of the variable target function method, employing methods of conjugate gradients and molecular dynamics with simulated annealing. Several slightly different models can be calculated by varying the initial structure. [1] ~ali, A. and Blundell, T.L. (1993) Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779-815. [2] gali, A. and Overington, J.P. (1994) Derivation of rules for comparative protein modeling from a database of protein structure alignments. Prot. Sci. 3, 1582-1596. [3] gali, A. (1995) Modelling mutations and homologous proteins. Curr. Opin. Biotech 6, 437-451. [4] gala, A., Potterton, L., Yuan, F., van Vlijmen, H. and Karplus, M. Evaluation of comparative protein modelling by MODELLER. Protein, in press. Residues near the amino termini contribute to hapten recognition in a cloned anti-17p-estradiol Fab fragment.
Petri Saviranta, Piitu Jauria, Maria Pajunen, Urpo Lamminmfiki, Timo LSvgen, Department of Biotechnology, University
of Turku, FIN-20520 Turku, Finland. We have cloned and sequenced the Fd and kappa chains of a monoclonal antibody against 17fl-estradiol is order to study the binding site at the molecular level. The chains were cloned into the vector pComb3 [1] which was used both for Fab production and for phage display in E. coll. The cloning sites in the vector are designed for the in-frame fusion of the amino termini of the mature Fd and light chains to pelB signal sequences, to enable secretion to periplasm where Fab assembly occurs. The integrity of the recombinant Fab was assessed by comparison of its binding characteristics to a proteolytic Fab fragment of the parental hybridoma IgG. The affinity for estradiol was 2-fold decreased and cross-reactivity to testosterone was 3.5-fold increased. Since the cloning sites for the Fd and the light chain in the pComb3 vector were embedded in the framework 1 coding sequence, the amino termini of the chains were not strictly authentic. Keeping this as a tentative explanation for the observed changes in steroid binding, we
removed the embedded cloning sites to restore the original coding sequences. The revised Fab showed binding properties identical with the proteolytic fragment, confirming that the observed differences resulted from changes in the amino termini. Furthermore, restoration of the heavy chain alone affected for estradiol while the restoration of the heavy chain alone affected only testosterone cross-reactivity. The restoration of the N-Terminal sequences also improved the Fab yield and immunoreactivity by a factor of 3, primarily attributed to the influence of the heavy chain N-terminus. We have shown that the very first N-terminal residues of the antibody are able to modify hapten binding, despite the fact that they cannot be in direct contact with the cleft-bound hapten. [1] Barbas et al. (1991) Proc. Nat. Acad. Sci. USA 88, 797882) Bacterial expression of a human Fv fragment IgM with a novel antibody combining site.
Grant I. Shoebridge, Robert L. Raison, Department of Cell and Molecular Biology, Immunobiology Unit, University of Technology Sydney, Australia. The third complementarity determining region of the heavy chain (HCDR3) is a major contributor to the architecture of the antibody combining site and hence the antigen binding specificity of an immunoglobulin molecule. We have previously described an unusual HCDR3 structure from a human IgM paraprotein Pot, obtained from a patient with Waldenstrom's macroglobulinemia [1]. Pot exhibits ployreactive properties binding to several structurally unrelated antigens. Crystallographic data indicates the Pot, Fv does not have a binding site shaped like a pocket or a groove. The HCDR3 fills the space between the VH/VL interface, blocking any binding site cavity. POT Fv may therefore serve as a model system for studying thr correlation between the HCDR3 primary amino acid sequence, three dimensional structure and polyreactive binding properties. In order to express Pot Fv, a dicistronic expression vector has been constructed. Secretion signals Omp A and Pel B direct export of the VH and VL into the bacterial periplasm respectively. Optimisation of expression conditions produced yields of Img/L isolated from the culture supernatant CDR grafting experiments and site directed mutagenesis of the HCDR3 will allow identification of the major structural correlates the polyrcactivity. Comparison of the structural and binding properties of the mutagenised POT Fv with native Pot Fv will enhance our understanding of the topology of the antibody combining site, particularly with respect to polyreactivity [1] Fan, Z-C et al. (1992) J. Mol. Biol. 228, 188. Effects of mutations in the heavy chain CDR3 region on the affinity of an anti-PSA diabody.
Liming Shu, Roger Smith, Ralph Abraham, John Link, Colleen Venti, Michael Darsley, JGEN, Inc. 16020 Industrial
Drive, Gaithersburg, MD 20877, USA. Prostate specific antigen (PSA) is a 33 KD glycoprotein of a single peptide chain. It is produced exclusively by prostate