Browsing by Subject "mRNA display"

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    Developing methods to understand and engineer protease cleavage specificity
    (2016-09) Lane, Michael
    Proteases are ubiquitous enzymes that comprise nearly 2% of all human genes. These robust enzymes are attractive potential therapeutics due to their catalytic turnover and capability for exquisite specificity. While most existing drugs require a stoichiometric ratio to function, therapeutic proteases could clear their targets much more efficiently. Unfortunately, existing technologies are inadequate for understanding and engineering therapeutic proteolytic specificities. My thesis work has focused on building the groundwork to enable these technologies to thrive. For the goal of engineering a new protease, it is currently necessary to identify prototype proteases for engineering efforts that have specificities similar to the desired target substrate. Current technologies are unable to characterize proteases adequately for this goal. Accordingly, I invested in developing a method for the accurate characterization of protease cleavage specificity. Our unique combination of mRNA display technology, Next-Generation Sequencing, and mass spectrometry enables the sampling of all possible permutations of octamer substrates and the identification of millions of cleavage sites. The throughput of our approach is orders of magnitude greater than the current state-of-the-art methods. The resulting high-resolution specificity maps can be applied to identify promising protease prototypes, predict human cross-reactivity, or lead to a better understanding of this critical component of natural physiology. In the work presented here, I applied my new specificity-screening method to assess the specificities of the proteases factor Xa, ADAM17, and streptopain. The resulting cleavage preference maps confirmed known specificities, and revealed new insight into the broad preferences of both narrow- and broad-specificity proteases. In particular, disfavored amino acids were illuminated better than ever before. The next focus of my work was to engineer multiple-subsite novel protease specificity. I chose streptopain as the prototype for my efforts to neutralize the superantigen exotoxin SpeA. I identified a target loop of SpeA wherein cleavage would result in inactive fragments. Further, I confirmed that streptopain can be successfully presented as an mRNA displayed fusion. In summary, my thesis work established crucial methodologies for applying mRNA display technology to enable the understanding and ultimately engineering the specificity of therapeutic proteases.
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    In vitro evolution of artificial enzymes: method development and applications
    (2014-09) Haugner III, John Christian
    Artificial enzymes have the potential to aid in the production of pharmaceuticals and facilitate basic biomedical research. There are two methods for making artificial enzymes: rational design and de novo selection. Rational design utilizes detailed knowledge of enzyme catalysis to design an enzyme active site, and then introduces this active site into a protein. However, due to the limited understanding of protein folding and structure-function relationships this approach is still extremely challenging and far from routine. In contrast, we utilize a directed evolution approach to isolate de novo artificial enzymes from a large library of protein variants by in vitro selection. Each of the trillions of proteins in a library are tested in a single experiment to determine if any have the desired activity. The artificial enzymes are created when the library is made so a high quality library is important for success. My thesis research focuses on two goals: (1) Construct a library built on the robust (alpha/beta)8 barrel enzyme scaffold for future enzyme selections and (2) Characterize a thermostable artificial RNA ligase and develop an application for this enzyme. The (alpha/beta)8 fold is used to catalyze a wide range of chemical reactions in nature. We used this fold to create a library containing > 1014 unique proteins by replacing loops of the catalytic face with randomized codons via PCR. Small sub-libraries were subjected to a protease-based folding selection to improve library quality by enriching for folded sequences. The final folding-enriched library contained > 1012 folded proteins representing an up to 50-fold improvement relative to a control library. These libraries will provide a valuable source of new enzymes for future in vitro selections. The previously generated artificial RNA ligases join 5'-triphosphate RNA to the 3'-hydroxyl of a second RNA substrate; a reaction not observed in nature. However the enzymes were also highly dynamic, which prevented the solving of the protein structure by NMR or X-ray crystallography. A more structured enzyme, called ligase 10C, was isolated by performing the ligase selection at 65°C and its structure was solved revealing a novel primordial fold. Here, we describe the detailed biochemical characterization of ligase 10C. Using a variety of RNA substrates, we also determined how ligation rates change with sequence composition revealing an enzyme with broad sequence specificity. We developed a method for the specific ligation and sequencing of 5'-triphosphorylated RNA. These results highlight ligase 10C as an attractive tool for the selective isolation of 5'-triphosphate RNA from a complex mixture, something which is difficult with current methods.