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

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.

Expression of Fatty Acyl-CoA Ligase Drives One-Pot De Novo Synthesis of Membrane-Bound Vesicles in a Cell-Free Transcription-Translation System

“…For these reasons, we became interested in exploring simpler strategies for membrane lipid synthesis that would utilize soluble biochemical elements and a minimal number of chemical steps. Simplified noncanonical lipid membrane generation would facilitate future integration with bottom-up synthetic biology applications such as the creation of proteoliposomes and cell-free bioreactors. , …”

“…Simplified noncanonical lipid membrane generation would facilitate future integration with bottom-up synthetic biology applications such as the creation of proteoliposomes and cellfree bioreactors. 6,16 Here, we describe a novel lipid synthesizing system by repurposing the activity of fatty acyl-CoA ligases (FACLs). In particular, we utilized a mycobacterial FACL, FadD2, 17 to catalyze the formation of fatty acyl-CoAs (FA-CoA) (Figure 1B).…”

A low-cost, thermostable, cell-free protein synthesis platform for on demand production of conjugate vaccines

“…Interestingly, the CLM24 ΔIpxM performed better with the HEPES buffer at pH 7.2 (Figure S5) and 60 mg/mL maltodextrin appeared to have a detrimental impact on sfGFP yields with ~70% protein produced in the PEP MD formulation and ~50% protein produced in both the MD and MD min formulations compared to the original (PEP) formulation (Figure S3B). Despite this difference, the MD min formulation has a significantly lower cost per milliliter (Figure 2B and 2C) and enables protein yields sufficient for glycoconjugate vaccine production (~100 µg/mL) 50 , with a maximum yield of ~350 µg/mL sfGFP in the iVAX strain.…”

“…Lyophilized reactions behaved similarly under elevated temperatures when producing sfGFP (Figure 3) and PD (Figure 4A-C). Glycosylation was initiated after 4 hours of protein synthesis (~100-200 µg/mL of carrier protein produced) (Figure S9A), yielding > 57 µg/mL of glycosylated PD in all conditions before The copyright holder for this preprint this version posted August 12, 2022. ; https://doi.org/10.1101/2022.08.10.503507 doi: bioRxiv preprint storage 50 . The glycosylation activity is retained in all formulations before storage (Figure S10A) and preserved after four weeks of storage at 50 ºC (Figure 4E, Figure S10C (uncropped)).…”

Nano-Carriers of COVID-19 Vaccines: The Main Pillars of Efficacy

“…In this platform, membrane-bound cargo expressed in live Escherichia coli comprises membrane vesicles. CFE nano-characterization pipeline can be a promising platform for vaccine development [ 82 ]. All current and future nano-carriers that transport the different active compounds must display delivery efficiency, cell targeting, materials safety and thermostability [ 82–84 ].…”