Biology subjects

Hershewe, J. M.

Publications and source records attributed to Hershewe, J. M..

4 recordsLinked to original sources

bioRxiv2022

Ribosome display of N-linked glycoproteins in cell-free extracts

Ribosome display is a powerful in vitro method for the selection and directed evolution of proteins expressed from combinatorial libraries. However, because ribosome display is typically performed with standard in vitro translation reagents, the ability to display proteins with complex post-translational modifications such as glycosylation is limited. To address this technological gap, here we developed a set of complementary methods for producing stalled ribosome complexes that displayed asparagine-linked (N-linked) glycoproteins in conformations amenable to downstream functional and glyco-structural interrogation. The ability to generate glycosylated ribosome-nascent chain (glycoRNC) complexes was enabled by integrating SecM-mediated translation arrest with methods for cell-free synthesis of N-glycoproteins. This integration yielded a novel capability for translating and displaying target proteins modified efficiently and site-specifically with different N-glycan structures. Moreover, the encoding mRNAs remained stably attached to stalled ribosomes both before and after biopanning, thereby providing the genotype- glycophenotype link between an arrested glycoprotein and its RNA message. We anticipate that our method will enable selection and evolution of N-linked glycoproteins with advantageous biological and biophysical properties.

synthetic biology↗

bioRxiv2020

Characterizing and controlling nanoscale self-assembly of suckerin-12

Structural proteins such as the "suckerins" present promising avenues for fabricating functional materials. Suckerins are a family of naturally occurring block copolymer-type proteins that comprise the sucker ring teeth of cephalopods and are known to self-assemble into supramolecular networks of nanoconfined {beta}-sheets. Here, we report characterization and controllable, nanoscale self-assembly of suckerin-12 (S12). We characterize impacts of salt, pH, and protein concentration on S12 solubility, secondary structure, and self-assembly. In doing so, we identify conditions for fabricating ~100 nm nanoassemblies (NAs) with narrow size distributions. Finally, by installing a non-canonical amino acid (ncAA) into S12, we demonstrate the assembly of NAs that are covalently conjugated with a hydrophobic fluorophore, and the ability to change self-assembly and {beta}-sheet content by PEGylation. This work presents new insights into the biochemistry of suckerin-12 and demonstrates how ncAAs can be used to expedite and fine-tune the design of protein materials.

bioengineering↗

bioRxiv2020

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for accelerating the design of cellular function, on-demand biomanufacturing, portable diagnostics, and educational kits. Many essential biological processes that could endow CFE systems with desired functions, such as protein glycosylation, rely on the activity of membrane-bound components. However, without the use of synthetic membrane mimics, activating membrane-dependent functionality in bacterial CFE systems remains largely unstudied. Here, we address this gap by characterizing native, cell-derived membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membranebound machinery. We first use nanocharacterization techniques to show that lipid vesicles in CFE extracts are tens to hundreds of nanometers across, and on the order of ~3x1012 particles/mL. We then determine how extract processing methods, such as post-lysis centrifugation, can be used to modulate concentrations of membrane vesicles in CFE systems. By tuning these methods, we show that increasing the number of vesicle particles to ~7x1012 particles/mL can be used to increase concentrations of heterologous membrane protein cargo expressed prior to lysis. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving N-linked and O-linked glycoprotein synthesis. We anticipate that our findings will facilitate in vitro gene expression systems that require membrane-dependent activities and open new opportunities in glycoengineering.

synthetic biology↗

bioRxiv2019

Apparent size and morphology of bacterial microcompartments varies with technique

Bacterial microcompartments (MCPs) are protein-based organelles which encapsulate metabolic pathways. Metabolic engineers have recently sought to repurpose MCPs to encapsulate heterologous pathways to increase flux through pathways of interest. As MCP engineering becomes more common, standardized methods for analyzing changes to MCPs and interpreting results across studies will become increasingly important. In this study, we demonstrate that different imaging techniques yield variations in the apparent size of purified MCPs from Salmonella enterica serovar Typhimurium LT2, likely due to variations in sample preparation methods. We provide guidelines for preparing samples for MCP imaging and outline expected variations in apparent size and morphology between methods. With this report we aim to establish an aid for comparing results across studies.

synthetic biology↗