Subject(s)
Colloids , Magnetics , Proteins/chemistry , Fluorescence , Microscopy, Electron , Protein BindingABSTRACT
Results of the single molecule force spectroscopy study of specific interactions between ribonuclease barnase and its inhibitor barstar are presented. Experimental data obtained for the force loading rate ranging 2-70 nN/s are well approximated by a single straight line, from which the dissociation barrier of the width of 0.12 nm and height of 0.75-0.85 × 10(-19)J can be inferred. The measured value of specific interaction does not depend on the NaCl concentration. This apparently contradicts the well-known dependence of the binding energy of this pair on the salt concentration, but such a "contradiction" is explained by the insensitivity of the force spectroscopy data to the relatively long-range electrostatic interaction. The latter essentially contributes to the value of barnase-barstar binding energy revealed by biochemical measurements, and it is exactly this electrostatic interaction which is influenced by the salt concentration.
Subject(s)
Bacterial Proteins/metabolism , Microscopy, Atomic Force/methods , Protein Interaction Mapping/methods , Ribonucleases/metabolism , Bacterial Proteins/chemistry , Microscopy, Atomic Force/instrumentation , Models, Theoretical , Osmolar Concentration , Protein Binding , Protein Interaction Mapping/instrumentation , Ribonucleases/chemistry , Static Electricity , Substrate SpecificityABSTRACT
The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high affinity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human tumors) was attached through a flexible linker to the second exon of human antibodies of IgG1 or IgE isotypes constant gene. The humanized construct of IgE isotype was generated for the first time. Genes of the recombinant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) promoter. Transfected HEK-293 cells efficiently produced antibodies of the corresponding isotypes IgE and IgG1. The results of Western blotting confirmed homogeneity of the expressed antibodies, which had the predicted molecular weight and specifically interacted with the HER-2/neu. The attachment of leader peptide to the 5'-end of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These results indicate that de novo constructed humanized immunoglobulin genes express functionally active, single-chain recombinant antibodies in eukaryotic cells.
Subject(s)
Antibodies, Neoplasm/biosynthesis , Immunoglobulin E/biosynthesis , Immunoglobulin G/biosynthesis , Immunoglobulin Variable Region/biosynthesis , Receptor, ErbB-2/immunology , Recombinant Proteins/biosynthesis , Antibodies, Neoplasm/genetics , Antibodies, Neoplasm/immunology , Cell Line , Gene Expression , Humans , Immunoglobulin E/genetics , Immunoglobulin E/immunology , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Receptor, ErbB-2/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/immunologyABSTRACT
The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high affinity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human tumors) was attached through a flexible linker to the second exon of human antibodies of IgG or IgE isotypes constant gene. The humanized construct of IgE isotype was generated for the first time. Genes of the recombinant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) promoter. Transfected HEK-293 cells efficiently produced antibodies of the corresponding isotypes IgE and IgG1. The results of Western blotting confirmed homogeneity of the expressed antibodies, which had the predicted molecular weight and specifically interacted with the HER-2/neu. The attachment of leader peptide to the 5'-end of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These results indicate that de novo constructed humanized immunoglobulin genes express functionally active, single-chain recombinant antibodies in eukaryotic cells.
