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

Jasmine M. Hershewe, Katherine F. Warfel, Shaelyn M. Iyer, Justin A. Peruzzi, Claretta J. Sullivan, Eric W. Roth, Matthew P. DeLisa, Neha P. Kamat and Michael C. Jewett ()
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Jasmine M. Hershewe: Northwestern University, Technological Institute E136
Katherine F. Warfel: Northwestern University, Technological Institute E136
Shaelyn M. Iyer: Northwestern University, Technological Institute E136
Justin A. Peruzzi: Northwestern University, Technological Institute E136
Claretta J. Sullivan: Wright-Patterson Air Force Base
Eric W. Roth: Tech Institute A/B Wing A173
Matthew P. DeLisa: Cornell University
Neha P. Kamat: Northwestern University
Michael C. Jewett: Northwestern University, Technological Institute E136

Nature Communications, 2021, vol. 12, issue 1, 1-12

Abstract: Abstract Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

Date: 2021
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DOI: 10.1038/s41467-021-22329-3

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