@article{hussain_lockney_wang_gera_rao_2013, title={Avidity-mediated virus separation using a hyperthermophilic affinity ligand}, volume={29}, ISSN={["8756-7938"]}, DOI={10.1002/btpr.1655}, abstractNote={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 (KD ∼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. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013}, number={1}, journal={BIOTECHNOLOGY PROGRESS}, author={Hussain, Mahmud and Lockney, Dustin and Wang, Ruqi and Gera, Nimish and Rao, Balaji M.}, year={2013}, pages={237–246} }
 @misc{gera_hussain_rao_2013, title={Protein selection using yeast surface display}, volume={60}, ISSN={["1046-2023"]}, DOI={10.1016/j.ymeth.2012.03.014}, abstractNote={Binding proteins are typically isolated from combinatorial libraries of scaffold proteins using one of the many library screening tools available, such as phage display, yeast surface display or mRNA display. A key principle underlying these screening technologies is the establishment of a link between each unique mutant protein and its corresponding genetic code. The mutant proteins binding a desired target species are separated and subsequently identified using the genetic code. In this review, we largely focus on the use of yeast surface display for the isolation of binding proteins from combinatorial libraries. In yeast surface display, the yeast cell links the mutant protein to its coding DNA. Each yeast cell expresses the mutant proteins as fusions to a yeast cell wall protein; the yeast cell also carries plasmid DNA that codes for the mutant protein. Over the years, the yeast surface display platform has emerged as a powerful tool for protein engineering, and has been used in a variety of applications including affinity maturation, epitope mapping and biophysical characterization of proteins. Here we present a broad overview of the yeast surface display system and its applications, and compare it with other contemporary screening platforms. Further, we present detailed protocols for the use of yeast surface display to isolate de novo binding proteins from combinatorial libraries, and subsequent biophysical characterization of binders. These protocols can also be easily modified for affinity maturation of the isolated de novo binders.}, number={1}, journal={METHODS}, author={Gera, Nimish and Hussain, Mahmud and Rao, Balaji M.}, year={2013}, month={Mar}, pages={15–26} }
 @article{hussain_gera_hill_rao_2013, title={Scaffold Diversification Enhances Effectiveness of a Super library of Hyperthermophilic Proteins}, volume={2}, ISSN={["2161-5063"]}, DOI={10.1021/sb300029m}, abstractNote={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 "superlibrary" or "library-of-libraries" 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.}, number={1}, journal={ACS SYNTHETIC BIOLOGY}, author={Hussain, Mahmud and Gera, Nimish and Hill, Andrew B. and Rao, Balaji M.}, year={2013}, month={Jan}, pages={6–13} }
 @article{menegatti_hussain_naik_carbonell_rao_2013, title={mRNA display selection and solid-phase synthesis of Fc-binding cyclic peptide affinity ligands}, volume={110}, DOI={10.1002/bit.24760}, abstractNote={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. Biotechnol. Bioeng. 2013; 110: 857–870. © 2012 Wiley Periodicals, Inc.}, number={3}, journal={Biotechnology and Bioengineering}, author={Menegatti, S. and Hussain, M. and Naik, A. D. and Carbonell, R. G. and Rao, B. M.}, year={2013}, pages={857–870} }
 @article{gera_hussain_wright_rao_2011, title={Highly Stable Binding Proteins Derived from the Hyperthermophilic Sso7d Scaffold}, volume={409}, ISSN={["0022-2836"]}, DOI={10.1016/j.jmb.2011.04.020}, abstractNote={We have shown that highly stable binding proteins for a wide spectrum of targets can be generated through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Sso7d is a small (∼ 7 kDa, 63 amino acids) DNA-binding protein that lacks cysteine residues and has a melting temperature of nearly 100 °C. We generated a library of 108 Sso7d mutants by randomizing 10 amino acid residues on the DNA-binding surface of Sso7d, using yeast surface display. Binding proteins for a diverse set of model targets could be isolated from this library; our chosen targets included a small organic molecule (fluorescein), a 12 amino acid peptide fragment from the C-terminus of β-catenin, the model proteins hen egg lysozyme and streptavidin, and immunoglobulins from chicken and mouse. Without the application of any affinity maturation strategy, the binding proteins isolated had equilibrium dissociation constants in the nanomolar to micromolar range. Further, Sso7d-derived binding proteins could discriminate between closely related immunoglobulins. Mutant proteins based on Sso7d were expressed at high yields in the Escherichia coli cytoplasm. Despite extensive mutagenesis, Sso7d mutants have high thermal stability; five of six mutants analyzed have melting temperatures > 89 °C. They are also resistant to chemical denaturation by guanidine hydrochloride and retain their secondary structure after extended incubation at extreme pH values. Because of their favorable properties, such as ease of recombinant expression, and high thermal, chemical and pH stability, Sso7d-derived binding proteins will have wide applicability in several areas of biotechnology and medicine.}, number={4}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Gera, Nimish and Hussain, Mahmud and Wright, Robert C. and Rao, Balaji M.}, year={2011}, month={Jun}, pages={601–616} }