The ability to create affinity reagents that specifically bind to (or recognize) a molecular targ... more The ability to create affinity reagents that specifically bind to (or recognize) a molecular target is critical for applied biotechnology as well as fundamental research in biology. For instance, the specific recognition of targets present in complex mixtures is a key step in the detection and/or quantification of molecular species. Examples include detection of disease biomarkers in blood or trace explosives in water, and measurement of protein concentrations in cell lysates for quantitative systems biology. Antibodies are the most commonly used affinity reagents. Antibodies, however, are expensive to produce and typically have low stability. Also, the generation of antibodies for new targets through the frequently used procedure of immunizing animals, is a lengthy and tedious process. As an alternative to antibodies, we are investigating the use of small protein domains from extremophilic organisms as scaffolds or templates for generating affinity reagents. To this end, we have id...
Antibodies are the most commonly used binding molecules in biomolecular measurement. However, ant... more Antibodies are the most commonly used binding molecules in biomolecular measurement. However, antibodies have several disadvantages such as low thermodynamic stability, a large multi-domain structure stabilized by disulfide bonds and high cost of production. As an alternative to antibodies, we are investigating the use of a set of small protein domains from extremophilic organisms as novel scaffolds for engineering molecular recognition, i.e. a template for generating protein variants that bind with high affinity and specificity to target species. As part of this effort, we have identified and cloned multiple small protein domains from the hyperthermophilic organisms Thermotoga maritima, Sulfolobus solfataricus and Pyrococcus furiosus. The characteristic features of these domains are: (i) high thermal stability (ii) lack of cysteines; and (iii) low molecular weight (< ~10-12 kDa). Using a combination of yeast surface display and mRNA display, we are currently screening large libr...
The use of binding proteins from non-immunoglobulin scaffolds has become increasingly common in b... more The use of binding proteins from non-immunoglobulin scaffolds has become increasingly common in biotechnology and medicine. Typically, binders are isolated from a combinatorial library generated by mutating a single scaffold protein. In contrast, here we generated a &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;superlibrary&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; or &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;library-of-libraries&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; of 4 × 10(8) protein variants by mutagenesis of seven different hyperthermophilic proteins; six of the seven proteins have not been used as scaffolds prior to this study. Binding proteins for five different model targets were successfully isolated from this library. Binders obtained were derived from five out of the seven scaffolds. Strikingly, binders from this modestly sized superlibrary have affinities comparable or higher than those obtained from a library with 1000-fold higher sequence diversity but derived from a single stable scaffold. Thus scaffold diversification, i.e., randomization of multiple different scaffolds, is a powerful alternate strategy for combinatorial library construction.
Immunoaffinity separation of large multivalent species such as viruses is limited by the stringen... more Immunoaffinity separation of large multivalent species such as viruses is limited by the stringent elution conditions necessary to overcome their strong and highly avid interaction with immobilized affinity ligands on the capture surface. Here we present an alternate strategy that harnesses the avidity effect to overcome this limitation. Red clover necrotic mosaic virus (RCNMV), a plant virus relevant to drug delivery applications, was chosen as a model target for this study. An RCNMV binding protein (RBP) with modest binding affinity (K(D) ~100 nM) was generated through mutagenesis of the Sso7d protein from Sulfolobus solfataricus and used as the affinity ligand. In our separation scheme, RCNMV is captured by a highly avid interaction with RBP immobilized on a nickel surface through a hexahistidine (6xHis) tag. Subsequently, disruption of the multivalent interaction and release of RCNMV is achieved by elution of RBP from the nickel surface. Finally, RCNMV is separated from RBP by exploiting the large difference in their molecular weights (~8 MDa vs. ~10 kDa). Our strategy not only eliminates the need for harsh elution conditions, but also bypasses chemical conjugation of the affinity ligand to the capture surface. Stable non-antibody affinity ligands to a wide spectrum of targets can be generated through mutagenesis of Sso7d and other hyperthermophilic proteins. Therefore, our approach may be broadly relevant to cases where capture of large multivalent species from complex mixtures and subsequent release without the use of harsh elution conditions is necessary.
Cyclic peptides are attractive candidates for synthetic affinity ligands due to their favorable p... more Cyclic peptides are attractive candidates for synthetic affinity ligands due to their favorable properties, such as resistance to proteolysis, and higher affinity and specificity relative to linear peptides. Here we describe the discovery, synthesis and characterization of novel cyclic peptide affinity ligands that bind the Fc portion of human Immunoglobulin G (IgG; hFc). We generated an mRNA display library of cyclic pentapeptides wherein peptide cyclization was achieved with high yield and selectivity, using a solid-phase crosslinking reaction between two primary amine groups, mediated by a homobifunctional linker. Subsequently, a pool of cyclic peptide binders to hFc was isolated from this library and chromatographic resins incorporating the selected cyclic peptides were prepared by on-resin solid-phase peptide synthesis and cyclization. Significantly, this approach results in resins that are resistant to harsh basic conditions of column cleaning and regeneration. Further studies identified a specific cyclic peptide--cyclo[Link-M-WFRHY-K]--as a robust affinity ligand for purification of IgG from complex mixtures. The cyclo[Link-M-WFRHY-K] resin bound selectively to the Fc fragment of IgG, with no binding to the Fab fragment, and also bound immunoglobulins from a variety of mammalian species. Notably, while the recovery of IgG using the cyclo[Link-M-WFRHY-K] resin was comparable to a Protein A resin, elution of IgG could be achieved under milder conditions (pH 4 vs. pH 2.5). Thus, cyclo[Link-M-WFRHY-K] is an attractive candidate for developing a cost-effective and robust chromatographic resin to purify monoclonal antibodies (mAbs). Finally, our approach can be extended to efficiently generate and evaluate cyclic peptide affinity ligands for other targets of interest.
The ability to create affinity reagents that specifically bind to (or recognize) a molecular targ... more The ability to create affinity reagents that specifically bind to (or recognize) a molecular target is critical for applied biotechnology as well as fundamental research in biology. For instance, the specific recognition of targets present in complex mixtures is a key step in the detection and/or quantification of molecular species. Examples include detection of disease biomarkers in blood or trace explosives in water, and measurement of protein concentrations in cell lysates for quantitative systems biology. Antibodies are the most commonly used affinity reagents. Antibodies, however, are expensive to produce and typically have low stability. Also, the generation of antibodies for new targets through the frequently used procedure of immunizing animals, is a lengthy and tedious process. As an alternative to antibodies, we are investigating the use of small protein domains from extremophilic organisms as scaffolds or templates for generating affinity reagents. To this end, we have id...
Antibodies are the most commonly used binding molecules in biomolecular measurement. However, ant... more Antibodies are the most commonly used binding molecules in biomolecular measurement. However, antibodies have several disadvantages such as low thermodynamic stability, a large multi-domain structure stabilized by disulfide bonds and high cost of production. As an alternative to antibodies, we are investigating the use of a set of small protein domains from extremophilic organisms as novel scaffolds for engineering molecular recognition, i.e. a template for generating protein variants that bind with high affinity and specificity to target species. As part of this effort, we have identified and cloned multiple small protein domains from the hyperthermophilic organisms Thermotoga maritima, Sulfolobus solfataricus and Pyrococcus furiosus. The characteristic features of these domains are: (i) high thermal stability (ii) lack of cysteines; and (iii) low molecular weight (< ~10-12 kDa). Using a combination of yeast surface display and mRNA display, we are currently screening large libr...
The use of binding proteins from non-immunoglobulin scaffolds has become increasingly common in b... more The use of binding proteins from non-immunoglobulin scaffolds has become increasingly common in biotechnology and medicine. Typically, binders are isolated from a combinatorial library generated by mutating a single scaffold protein. In contrast, here we generated a &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;superlibrary&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; or &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;library-of-libraries&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; of 4 × 10(8) protein variants by mutagenesis of seven different hyperthermophilic proteins; six of the seven proteins have not been used as scaffolds prior to this study. Binding proteins for five different model targets were successfully isolated from this library. Binders obtained were derived from five out of the seven scaffolds. Strikingly, binders from this modestly sized superlibrary have affinities comparable or higher than those obtained from a library with 1000-fold higher sequence diversity but derived from a single stable scaffold. Thus scaffold diversification, i.e., randomization of multiple different scaffolds, is a powerful alternate strategy for combinatorial library construction.
Immunoaffinity separation of large multivalent species such as viruses is limited by the stringen... more Immunoaffinity separation of large multivalent species such as viruses is limited by the stringent elution conditions necessary to overcome their strong and highly avid interaction with immobilized affinity ligands on the capture surface. Here we present an alternate strategy that harnesses the avidity effect to overcome this limitation. Red clover necrotic mosaic virus (RCNMV), a plant virus relevant to drug delivery applications, was chosen as a model target for this study. An RCNMV binding protein (RBP) with modest binding affinity (K(D) ~100 nM) was generated through mutagenesis of the Sso7d protein from Sulfolobus solfataricus and used as the affinity ligand. In our separation scheme, RCNMV is captured by a highly avid interaction with RBP immobilized on a nickel surface through a hexahistidine (6xHis) tag. Subsequently, disruption of the multivalent interaction and release of RCNMV is achieved by elution of RBP from the nickel surface. Finally, RCNMV is separated from RBP by exploiting the large difference in their molecular weights (~8 MDa vs. ~10 kDa). Our strategy not only eliminates the need for harsh elution conditions, but also bypasses chemical conjugation of the affinity ligand to the capture surface. Stable non-antibody affinity ligands to a wide spectrum of targets can be generated through mutagenesis of Sso7d and other hyperthermophilic proteins. Therefore, our approach may be broadly relevant to cases where capture of large multivalent species from complex mixtures and subsequent release without the use of harsh elution conditions is necessary.
Cyclic peptides are attractive candidates for synthetic affinity ligands due to their favorable p... more Cyclic peptides are attractive candidates for synthetic affinity ligands due to their favorable properties, such as resistance to proteolysis, and higher affinity and specificity relative to linear peptides. Here we describe the discovery, synthesis and characterization of novel cyclic peptide affinity ligands that bind the Fc portion of human Immunoglobulin G (IgG; hFc). We generated an mRNA display library of cyclic pentapeptides wherein peptide cyclization was achieved with high yield and selectivity, using a solid-phase crosslinking reaction between two primary amine groups, mediated by a homobifunctional linker. Subsequently, a pool of cyclic peptide binders to hFc was isolated from this library and chromatographic resins incorporating the selected cyclic peptides were prepared by on-resin solid-phase peptide synthesis and cyclization. Significantly, this approach results in resins that are resistant to harsh basic conditions of column cleaning and regeneration. Further studies identified a specific cyclic peptide--cyclo[Link-M-WFRHY-K]--as a robust affinity ligand for purification of IgG from complex mixtures. The cyclo[Link-M-WFRHY-K] resin bound selectively to the Fc fragment of IgG, with no binding to the Fab fragment, and also bound immunoglobulins from a variety of mammalian species. Notably, while the recovery of IgG using the cyclo[Link-M-WFRHY-K] resin was comparable to a Protein A resin, elution of IgG could be achieved under milder conditions (pH 4 vs. pH 2.5). Thus, cyclo[Link-M-WFRHY-K] is an attractive candidate for developing a cost-effective and robust chromatographic resin to purify monoclonal antibodies (mAbs). Finally, our approach can be extended to efficiently generate and evaluate cyclic peptide affinity ligands for other targets of interest.
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