@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} }
 @article{goli_gera_liu_rao_rojas_genzer_2013, title={Generation and Properties of Antibacterial Coatings Based on Electrostatic Attachment of Silver Nanoparticles to Protein-Coated Polypropylene Fibers}, volume={5}, ISSN={["1944-8252"]}, DOI={10.1021/am4011644}, abstractNote={We present a simple method for attaching silver nanoparticles to polypropylene (PP) fibers in a two-step process to impart antibacterial properties. Specifically, PP fibers are pretreated by the adsorption from an aqueous solution of heat-denatured lysozyme (LYS) followed by LYS cross-linking using glutaraldehyde and sodium borohydride. At neutral pH, the surface of the adsorbed LYS layer is enriched with numerous positive charges. Silver nanoparticles (AgNPs) capped with trisodium citrate are subsequently deposited onto the protein-coated PP. Nanoparticle binding is mediated by electrostatic interactions between the positively charged LYS layer and the negatively charged AgNPs. The density of AgNPs deposited on PP depends on the amount of protein adsorbed on the surface. UV-vis spectroscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and scanning electron microscopy are employed to follow all preparation steps and to characterize the resulting functional surfaces. The antibacterial activity of the modified surfaces is tested against gram negative bacteria Escherichia coli (E. coli). Overall, our results show that PP surfaces coated with AgNPs exhibit excellent antibacterial activity with 100% removal efficiency.}, number={11}, journal={ACS APPLIED MATERIALS & INTERFACES}, author={Goli, Kiran K. and Gera, Nimish and Liu, Xiaomeng and Rao, Balaji M. and Rojas, Orlando J. and Genzer, Jan}, year={2013}, month={Jun}, pages={5298–5306} }
 @article{canbolat_gera_tang_monian_rao_pourdeyhimi_khan_2013, title={Preservation of Cell Viability and Protein Conformation on Immobilization within Nanofibers via Electrospinning Functionalized Yeast}, volume={5}, ISSN={["1944-8252"]}, DOI={10.1021/am4022768}, abstractNote={We investigate the immobilization of a model system of functionalized yeast that surface-display enhanced green fluorescent protein (eGFP) within chemically crosslinked polyvinyl alcohol (PVA) nanofibers. Yeast is incorporated into water insoluble nanofibrous materials by direct electrospinning with PVA followed by vapor phase chemical crosslinking of the polymer. Incorporation of yeast into the fibers is confirmed by elemental analysis and the viability is indicated by live/dead staining. Following electrospinning and crosslinking, we confirm that the yeast maintains its viability as well as the ability to express eGFP in the correct conformation. This method of processing functionalized yeast may thus be a powerful tool in the direct immobilization of properly folded, active enzymes within electrospun nanofibers with potential applications in biocatalysis.}, number={19}, journal={ACS APPLIED MATERIALS & INTERFACES}, author={Canbolat, M. Fatih and Gera, Nimish and Tang, Christina and Monian, Brinda and Rao, Balaji M. and Pourdeyhimi, Behnam and Khan, Saad A.}, year={2013}, month={Oct}, pages={9349–9354} }
 @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{gera_hill_white_carbonell_rao_2012, title={Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold}, volume={7}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0048928}, abstractNote={We have engineered pH sensitive binding proteins for the Fc portion of human immunoglobulin G (hIgG) (hFc) using two different strategies – histidine scanning and random mutagenesis. We obtained an hFc-binding protein, Sso7d-hFc, through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus; Sso7d-hFc was isolated from a combinatorial library of Sso7d mutants using yeast surface display. Subsequently, we identified a pH sensitive mutant, Sso7d-his-hFc, through systematic evaluation of Sso7d-hFc mutants containing single histidine substitutions. In parallel, we also developed a yeast display screening strategy to isolate a different pH sensitive hFc binder, Sso7d-ev-hFc, from a library of mutants obtained by random mutagenesis of a pool of hFc binders. In contrast to Sso7d-hFc, both Sso7d-his-hFc and Sso7d-ev-hFc have a higher binding affinity for hFc at pH 7.4 than at pH 4.5. The Sso7d-mutant hFc binders can be recombinantly expressed at high yield in E. coli and are monomeric in solution. They bind an epitope in the CH3 domain of hFc that has high sequence homology in all four hIgG isotypes (hIgG1–4), and recognize hIgG1–4 as well as deglycosylated hIgG in western blotting assays. pH sensitive hFc binders are attractive candidates for use in chromatography, to achieve elution of IgG under milder pH conditions. However, the surface density of immobilized hFc binders, as well as the avidity effect arising from the multivalent interaction of dimeric hFc with the capture surface, influences the pH dependence of dissociation from the capture surface. Therefore, further studies are needed to evaluate if the Sso7d mutants identified in this study are indeed useful as affinity ligands in chromatography.}, number={11}, journal={PLOS ONE}, author={Gera, Nimish and Hill, Andrew B. and White, Dalon P. and Carbonell, Ruben G. and Rao, Balaji M.}, year={2012}, month={Nov} }
 @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} }