@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={Abstract}, 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={Abstract}, 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} }