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NUANCE: Nanoscale Characterization Experimental Center

Eric W. Roth, NUANCE’s BioCryo EM Specialist, Co-Authors Publication in Nature Communications

The article “Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles” published in Nature Communication on April 22, 2021, led by Jasmine Hershewe of the Jewett group here at Northwestern, is in part, about developing and streamlining new technologies for research and biotech, including new methods of improving the quality and method of production of membrane bound vesicles (microscopic spherical containers) produced from lysing (destroying) e. coli cells.

These vesicles are used for CFE (cell-free gene expression) and are of particular interest in the production of glycoproteins that are studied and used in immunology research, as well as in helping to manufacture and optimize vaccines. Traditional synthetic glycoprotein production results in very low yields at high cost, however “By applying our optimized methods to increase concentrations of vesicle-bound glycosylation machinery, we shorten the time associated with extract preparation, increase glycosylation efficiencies, and enhance glycoprotein titers (concentration in solution) by up to ~170%.”

As an Electron Microscopy Specialist in NUANCE’s BioCryo Facility, Eric Roth shares: “My end of the work focused on the optimization of sample prep methods related to characterizing these vesicles, as well as the actual characterization of vesicle morphology by cryo-electron microscopy (see Figure 2) in our BioCryo facility."

Eric Roth Publication screen shot
a DLS analysis of crude extracts (blue) and SEC purified vesicles (green). Crude extract data are presented as mean intensity values for a given size ± standard deviation (SD) of n = 9 measurements (3 biologically independent extracts, each measured 3 times). Purified vesicle data are presented as mean intensity values for a given size ± SD of n = 3 measurements (one purified fraction measured 3 times). b NTA of purified vesicles collected from SEC. Mean and mode diameters observed in the particle size distribution are listed in the inset. Data are presented as mean ± standard error of the mean (SEM) of n = 5, 1 min NTA measurements of purified vesicles. c Illustration of particles detected in crude CFE extracts. d Cryo-EM micrographs of crude extracts. Black arrows indicate vesicles with apparent unilamellar morphology. White arrows indicate nested or multilamellar morphologies. Cropped images indicate representative vesicles. Scale bars are 100 nm.