Browsing by Author "Motabar, Dana"

Now showing 1 - 3 of 3
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    THE DESIGN OF TWO PROTEINS THAT HAVE 100% SEQUENCE IDENTITY BUT ENCODE DIFFERENT FOLDS
    (2015) Motabar, Dana; Bryan, Philip; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    It is well-established that proteins adopt specific three-dimensional structures. However, examples of proteins that can adopt more than one folded state have become increasingly more common. The objective of this thesis is to determine how three common, small folds are connected in sequence space. The folds this work focuses on are a 3-α-helix bundle, an α/β plait, and a 4β+α fold. Topological alignment and site-directed mutagenesis were used to develop engineered variants of the 3-α-helix bundle and the α/β plait folds that maintain their highly distinct native folds even though their sequences are 100% identical. CD and NMR data suggest that both proteins were stable and folded. This engineered fold switch demonstrates that the fold preference of a sequence is dependent upon stabilizing interactions within the context of the protein. These fold switching proteins have important implications in areas such as protein design, human disease, and structural biology.
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    DEVELOPMENT OF AN ELECTROCHEMICAL-BASED TOOLKIT FOR IMPROVED BIOPROCESSING APPLICATIONS
    (2023) Motabar, Dana; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Recombinant antibody therapeutics have become an important class of biopharmaceuticals that have shown effectiveness in treating a variety of diseases such as cancer, infection, and inflammation due to their high binding affinity and specificity. Importantly, process conditions established during the development and manufacture of antibodies dramatically impacts their quality, clinical efficacy, and safety. For process monitoring and control purposes, analytical technologies that enable rapid and cost-effective assessment of therapeutics are needed as they trim development time and costs. To address this need, we developed electrochemical-based analytical technologies that will enable low volume, near real-time monitoring of product quality attributes and process parameters. First, we demonstrate the development of thiolated PEG-based sensor interfaces for the detection of antibody titer and N-linked glycosylation. The interfaces couple electrochemical techniques with molecular recognition-based elements and a novel spectroelectrochemical reporter to provide rapid assessment of titer and galactosylation. Next, we demonstrate successful integration of the sensor interfaces with a microfluidic device in order to enable rapid, low volume sampling that is amenable to on-line monitoring. Lastly, we apply a mediated electrochemical probing (MEP) approach that uses redox mediators to quantitatively characterize redox-based quality information of antibodies that have undergone reduction or oxidation events. We believe that these technologies can provide fast, quantifiable results for bioprocessing applications and offer advantages in their simplicity, rapid response, and connectivity to electronics.
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    Protein G: β-galactosidase fusion protein for multi-modal bioanalytical applications
    (Wiley, 2022-08-17) Motabar, Dana; Wang, Sally; Tsao, Chen-Yu; Payne, Gregory F.; Bentley, William E.
    β-galactosidase (β-gal) is one of the most prevalent markers of gene expression. Its activity can be monitored via optical and fluorescence microscopy, electrochemistry, and many other ways after slight modification using protein engineering. Here, we have constructed a chimeric version that incorporates a streptococcal protein G domain at the N-terminus of β-gal that binds immunoglobins, namely IgG. This protein G: β-galactosidase fusion enables β-gal-based spectrophotometric and electrochemical measurements of IgG. Moreover, our results show linearity over an industrially relevant range. We demonstrate applicability with rapid spectroelectrochemical detection of IgG in several formats including using an electrochemical sensing interface that is rapidly assembled directly onto electrodes for incorporation into biohybrid devices. The fusion protein enables sensitive, linear, and rapid responses, and in our case, makes IgG measurements quite robust and simple, expanding the molecular diagnostics toolkit for biological measurement.