Rapid biosynthesis of glycoprotein therapeutics and vaccines from freeze-dried bacterial cell lysates.

The advent of distributed biomanufacturing platforms promises to increase agility in biologic production and expand access by reducing reliance on refrigerated supply chains. However, such platforms are not capable of robustly producing glycoproteins, which represent the majority of biologics approved or in development. To address this limitation, we developed cell-free technologies that enable rapid, modular production of glycoprotein therapeutics and vaccines from freeze-dried Escherichia coli cell lysates.

Cell-Free Protein Synthesis of Particulate Methane Monooxygenase into Nanodiscs.

Particulate methane monooxygenase (pMMO) is a multi-subunit membrane metalloenzyme used by methanotrophic bacteria to convert methane to methanol. A major hurdle to studying pMMO is the lack of a recombinant expression system, precluding investigation of individual residues by mutagenesis and hampering a complete understanding of its mechanism. Here, we developed an lysate-based cell-free protein synthesis (CFPS) system that can be used to express pMMO in vitro in the presence of nanodiscs.

Ribosome Stalling of -Linked Glycoproteins in Cell-Free Extracts.

Ribosome display is a powerful method for selection and directed evolution of proteins expressed from combinatorial libraries. However, the ability to display proteins with complex post-translational modifications such as glycosylation is limited. To address this gap, we developed a set of complementary methods for producing stalled ribosome complexes that displayed asparagine-linked (-linked) glycoproteins in conformations amenable to downstream functional and glycostructural interrogation.

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 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.

On-demand biomanufacturing of protective conjugate vaccines.

Conjugate vaccines are among the most effective methods for preventing bacterial infections. However, existing manufacturing approaches limit access to conjugate vaccines due to centralized production and cold chain distribution requirements. To address these limitations, we developed a modular technology for in vitro conjugate vaccine expression (iVAX) in portable, freeze-dried lysates from detoxified, nonpathogenic Upon rehydration, iVAX reactions synthesize clinically relevant doses of conjugate vaccines against diverse bacterial pathogens in 1 hour.

Characterizing and Controlling Nanoscale Self-Assembly of Suckerin-12.

Structural proteins such as "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 β-sheets. Here, we report the characterization and controllable, nanoscale self-assembly of suckerin-12 (S12).

Cell-free systems for accelerating glycoprotein expression and biomanufacturing.

Protein glycosylation, the enzymatic modification of amino acid sidechains with sugar moieties, plays critical roles in cellular function, human health, and biotechnology. However, studying and producing defined glycoproteins remains challenging. Cell-free glycoprotein synthesis systems, in which protein synthesis and glycosylation are performed in crude cell extracts, offer new approaches to address these challenges.

Apparent size and morphology of bacterial microcompartments varies with technique.

Bacterial microcompartments (MCPs) are protein-based organelles that 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.

High-Throughput Synthesis and Analysis of Intact Glycoproteins Using SAMDI-MS.

High-throughput quantification of the post-translational modification of many individual protein samples is challenging with current label-based methods. This paper demonstrates an efficient method that addresses this gap by combining -based cell-free protein synthesis (CFPS) and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to analyze intact proteins. This high-throughput approach begins with polyhistidine-tagged protein substrates expressed from linear DNA templates by CFPS.

A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases.

Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation.

A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases.

Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of posttranslational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins, of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3.