TY - JOUR
T1 - Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles
AU - Hershewe, Jasmine M.
AU - Warfel, Katherine F.
AU - Iyer, Shaelyn M.
AU - Peruzzi, Justin A.
AU - Sullivan, Claretta J.
AU - Roth, Eric W.
AU - DeLisa, Matthew P.
AU - Kamat, Neha P.
AU - Jewett, Michael C.
N1 - Funding Information:
We acknowledge Jessica Stark and Weston Kightlinger for helpful discussions about CFGpS, Aravind Natarajan and Dominic Mills for helpful discussions about O-OSTs and their substrate specificities, Ashty Karim for helpful discussions on data visualization, Han Teng Wong and Charlotte Abrahamson for fruitful discussions regarding immu-nostaining, and Maddie DeWinter for helpful discussions on LC-MS/MS methods. We thank Markus Aebi for providing the hR6 serum and Jeff Tabor for providing the DNA encoding NarX. This work made use of the Mass Spectrometry facility of the Integrated Molecular Structure Education and Research Center (IMSERC) at Northwestern University and the BioCryo facility of Northwestern University’s NUANCE Center, which has received support from the SHyNE Resource (NSF ECCS-1542205), the IIN, and Northwestern’s MRSEC program (NSF DMR-1720139). We gratefully acknowledge support from the Defense Threat Reduction Agency Grant HDTRA1-15-10052/P00001, the National Science Foundation Grants 1936789 and 1844336, the Air Force Research Laboratory Center of Excellence Grant FA8650-15-2-5518, the Bill & Melinda Gates Foundation Grant OPP1217652, the David and Lucile Packard Foundation, and the Camille Dreyfus Teacher-Scholar Program. This project was also supported in part by fellowships awarded to J.M.H. (NDSEG-36373) and K.F.W. (ND-CEN-013-096) through the National Defense Science and Engineering (NDSEG) Fellowship Program, sponsored by the Air Force Research Laboratory, the Office of Naval Research, and the Army Research Office. J.M.H and J.A.P. thank the Ryan Fellowship awarded by Northwestern University. J.A.P. was supported by an NSF Graduate Research Fellowship. The U.S. Government is authorized to reproduce and distribute reprints for Governmental purposes notwithstanding any copyright notation thereon. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of the Defense Threat Reduction Agency, or the U.S. Government.
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/12/1
Y1 - 2021/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41467-021-22329-3
DO - 10.1038/s41467-021-22329-3
M3 - Article
C2 - 33888690
AN - SCOPUS:85104795724
SN - 2041-1723
VL - 12
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 2363
ER -