Design of solubly expressed miniaturized SMART MHCs

Proceedings of the National Academy of Sciences · 2026-01-02 · 2 citations

The precise recognition of specific peptide–major histocompatibility complex (pMHC) complexes by T cell receptors (TCRs) plays a key role in infectious disease, cancer, and autoimmunity. A critical step in many immunobiological studies is the identification of T cells expressing TCRs specific to a given pMHC antigen. However, the intrinsic instability of empty class-I MHCs limits their soluble expression in Escherichia coli and makes it very difficult to characterize even a small fraction of possible pMHC/TCR interactions. To overcome this limitation, we designed small proteins which buttress the peptide binding groove of class I MHCs, replacing β2-microglobulin (β2m) and the heavy chain α3 domain, and enable soluble and partially soluble expression in E. coli of H-2D b and A*02:01, respectively. We demonstrate that these soluble, monomeric, antigen-receptive, truncated (SMART) MHCs retain both peptide- and TCR-binding specificity and that peptide-bound structures of both allomorphs are similar to their full-length, native counterparts. With extension to the majority of HLA alleles, SMART MHCs should be broadly useful for probing the T cell repertoire in approaches ranging from yeast display to T cell staining.

Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression

Nature Communications · 2026-03-05 · 1 citations

Abstract Access to recombinant proteins is vital in basic science and biotechnology research. Cell-free gene expression systems provide one approach to address this need, but widespread utilization remains limited by the cost, complexity, and inconsistency of current platforms. To address these limitations, we carry out a multi-dimensional definitive screening design to reduce the number of reagent components and remove costly secondary energy substrates. From 1,231 different reagent formulations, we discover a simple and reproducible system based on 12 components. The optimized reagent formulation can produce 2.4 ± 0.3 g/L of protein product at the 15-µL scale (~$60/g protein ) and 3.7 ± 0.2 g/L (~$39/g protein ) at the 4-mL scale with oxygen supplementation. This provides an average 95% reduction in cost over previous cell-free reagent formulations. We further show that the optimized reagent formulation can produce nucleoside triphosphates from nitrogenous bases and ribose and that it is robust to failure across batches of cell lysates, users/locations, and in the synthesis of more than 20 different proteins. For example, we demonstrate the production of fifteen therapeutically relevant products, including full-length aglycosylated monoclonal antibodies. We anticipate that our optimized reagent formulation will democratize the use of cell-free systems for protein manufacturing and synthetic biology applications.

Impact of Process Interruptions in the Production of Lysates for Cell‐Free Expression Systems

Biotechnology and Bioengineering · 2026-04-05

Cell-free gene expression systems offer cell-like functionalities outside the confines of the cell, garnering increasing interest for applications from biomanufacturing to sensing. As applications expand, the need to implement economically scaled processes to produce cellular lysates grows. The protocols to produce these cellular lysates are complex, and the impact of altering many of the process variables remains understudied. Here, we set out to evaluate the effect of extended incubations at several points in the extract preparation process with the goal of identifying breakpoints that would enable flexibility in process implementation. As a model, we prepared lysates from 50 L cultures instead of typical 1 L volumes. We produced 72 lysates, 36 that were incubated overnight before and after culture centrifugation, and 36 that were incubated with and without a run-off reaction, each across different temperatures. We found that incubations before and after culture centrifugation substantially increased variability between culture replicates but did not reduce cell-free protein synthesis activity, contrary to conventional wisdom that materials should be kept cold as much as possible throughout the process. We also observed that omitting the run-off reaction reduced yields but resulted in lysates that were robust to incubation up to room temperature overnight. When a run-off reaction was included, activity dropped both as a function of duration and temperature, and the overall variability increased. Our work offers potential options for flexibility in implementing lysate production processes and motivates further investigation into how key processing steps relate to cell-free expression activity.

Topological reprogramming transforms an integral membrane oligosaccharyltransferase into a water-soluble glycosylation catalyst

bioRxiv (Cold Spring Harbor Laboratory) · 2026-01-31 · 1 citations

Abstract Glycosyltransferases (GTs) catalyze the formation of new glycosidic bonds and thus are vital for synthesizing nature’s vast repertoire of glycans and glycoconjugates and for engineering glycan-related medicines and materials. However, obtaining detailed structural and functional insights for the >750,000 known GTs is limited by difficulties associated with their efficient recombinant expression. Members of the GT-C fold, in particular, pose the most significant expression challenges due to the integration and folding requirements of their multiple membrane-spanning regions. Here, we address this challenge by engineering water-soluble variants of an archetypal GT-C fold enzyme, namely the oligosaccharyltransferase PglB from Campylobacter jejuni ( Cj PglB), which possesses 13 hydrophobic transmembrane helices. To render Cj PglB water-soluble, we leveraged two advanced protein engineering methods: one that is universal called SIMPLEx ( s olubilization of IMP s with high levels of ex pression) and the other that is custom tailored called WRAPs ( w ater-soluble R Fdiffused a mphipathic p roteins). Each approach was able to transform Cj PglB into a water-soluble enzyme that could be readily expressed in the cytoplasm of Escherichia coli cells at yields in the 3–6 mg/L range. Importantly, solubilization was achieved without the need for detergents and with retention of catalytic function. Collectively, our findings demonstrate that both SIMPLEx and WRAPs are promising platforms for advancing the molecular characterization of even the most structurally complex GTs, while also enabling broader GT-mediated glycosylation capabilities within synthetic glycobiology applications.

Design of solubly expressed miniaturized SMART MHCs

bioRxiv (Cold Spring Harbor Laboratory) · 2025-03-14 · 1 citations

Abstract The precise recognition of specific peptide-MHC (pMHC) complexes by T-cell receptors (TCRs) plays a key role in infectious disease, cancer and autoimmunity. A critical step in many immunobiological studies is the identification of T-cells expressing TCRs specific to a given pMHC antigen. However, the intrinsic instability of empty class-I MHCs limits their soluble expression in Escherichia coli ( E. coli ) and makes it very difficult to characterize even a small fraction of possible pMHC/TCR interactions. To overcome this limitation, we designed small proteins which buttress the peptide binding groove of class I MHCs, replacing β2-microglobulin (β2m) and the heavy chain α3 domain, and enable soluble expression of both H-2D b and A*02:01 in E. coli . We demonstrate that these soluble, monomeric, antigen-receptive, truncated (SMART) MHCs retain both peptide- and TCR-binding specificity, and that peptide-bound structures of both allomorphs are similar to their full-length, native counterparts. With extension to the majority of HLA alleles, SMART MHCs should be broadly useful for probing the T-cell repertoire in approaches ranging from yeast display to T-cell staining. Significance Despite the critical role that TCR/pMHC interactions play in human health, it has remained difficult to produce reagents necessary to study them. Requirements for refolding or sequence optimization limit immunologists’ and biochemists’ ability to characterize diverse pMHC/TCR interactions. Here, we develop a de-novo designed protein domain that stabilizes the H-2D b and A*02:01 class I MHC allomorphs, allowing soluble expression in E. coli without the need for a stabilizing peptide, and improving display on the yeast surface, while maintaining peptide and TCR binding interactions. These features facilitate a wide range of experiments to more fully understand the nature of pMHC/TCR interactions, and pave the way for the development of stabilizing domains for all MHC allomorphs.

Evaluation of Bacterial Microcompartment Cofactor Recycling and Permeability with a Model Guided <i>In Vitro</i> Assay

bioRxiv (Cold Spring Harbor Laboratory) · 2025-08-23 · 1 citations

Abstract Biomanufacturing is a promising strategy for sustainable chemical production. However, challenges such as cofactor competition and low pathway flux prevent competitive titers. Some bacteria address these challenges by encapsulating metabolic pathways in bacterial microcompartments (MCPs), many of which contain dedicated cofactor recycling enzymes. We sought to determine how pathway cofactor recycling and intermediate sequestration in MCPs benefit pathway performance using an in vitro assay and kinetic model of the 1,2-propanediol utilization (Pdu) system. Guided by model simulations, we performed experimental design to characterize permeability, a key and difficult-to-measure property of MCPs. Using our model and measurements of metabolite concentrations over time, we estimate MCP permeability values in the range of 10 −5 cm/s. We also demonstrated that NAD+/NADH recycling in the Pdu MCP benefits increased pathway flux. This study integrates experiments and systems modeling to advance our understanding of why pathways are encapsulated and to inform bioengineering applications.

Active learning-guided optimization of cell-free biosensors for lead testing in drinking water

bioRxiv (Cold Spring Harbor Laboratory) · 2025-08-20 · 1 citations

Point-of-use diagnostics based on allosteric transcription factors (aTFs) are promising tools for environmental monitoring and human health. However, biosensors relying on natural aTFs rarely exhibit the sensitivity and selectivity needed for real-world applications, and traditional directed evolution struggles to optimize multiple biosensor properties at once. To overcome these challenges, we develop a multi-objective, machine learning (ML)-guided cell-free gene expression workflow for engineering aTF-based biosensors. Our approach rapidly generates high-quality sequence-to-function data, which we transform into an augmented paired dataset to train an ML model using directional labels that capture how aTF mutations alter performance. We apply our workflow to engineer the aTF PbrR as a point-of-use diagnostic for lead contamination in water. We tune the sensitivity of PbrR to sense at the U.S. Environmental Protection Agency (EPA) action level for lead and modify the selectivity away from zinc, a common metal found in water supplies. Finally, we show that the engineered PbrR functions in freeze-dried cell-free reactions, enabling a diagnostic capable of detecting lead in drinking water down to ~5.7 ppb. Our ML-driven, multi-objective framework-powered by directional tokens-can generalize to other biosensors and proteins, accelerating the development of synthetic biology tools for biotechnology applications.

A Scalable Cell-Free Manufacturing Platform for Two-Step Bioproduction of Immunogenic Conjugate Vaccines

ACS Synthetic Biology · 2025-11-19

serotype 4 capsular polysaccharide, confirming the immunogenicity of the conjugate. We anticipate that this cell-free platform will advance efforts in decentralized manufacturing and rapid response to bacterial disease threats.

Can protein expression be ‘solved’?

Trends in biotechnology · 2025-06-02 · 15 citations

Recombinant protein expression is central to biotechnology's application. However, not all proteins can be expressed in all organisms, and, given the vast experimental space, it can be challenging to identify the conditions that will yield successful protein expression. The field lacks a predictive model of soluble protein expression that could replace laborious experimental trial and error. Here, we discuss the state of the field and identify the lack of large, high-fidelity datasets as the primary bottleneck to progress. We outline a proposed path toward an extensible experimental platform for collecting soluble overexpression data across organisms. We suggest that the resulting data should be used to train predictive models of protein expression toward answering the question: can protein expression be solved?

LDBT instead of DBTL: combining machine learning and rapid cell-free testing

Nature Communications · 2025-11-05 · 4 citations

Synthetic biology is defined by Design-Build-Test-Learn cycles. Recent advances in machine learning are changing the landscape; thus, we propose that “Learning” can precede “Design”. Moreover, adopting cell-free platforms can further accelerate “Building” and “Testing” for megascale data generation and models.