A Selection for Assembly Reveals That a Single Amino Acid Mutant of the Bacteriophage MS2 Coat Protein Forms a Smaller Virus-like Particle.

Virus-like particles are used to encapsulate drugs, imaging agents, enzymes, and other biologically active molecules in order to enhance their function. However, the size of most virus-like particles is inflexible, precluding the design of appropriately sized containers for different applications. Here, we describe a chromatographic selection for virus-like particle assembly. Using this selection, we identified a single amino acid substitution to the coat protein of bacteriophage MS2 that mediates a uniform switch in particle geometry from T = 3 to T = 1 icosahedral symmetry. The resulting smaller particle retains the ability to be disassembled and reassembled in vitro and to be chemically modified to load cargo into its interior cavity. The pair of 27 and 17 nm MS2 particles will allow direct examination of the effect of size on function in established applications of virus-like particles, including drug delivery and imaging.

In Vivo Site-Specific Protein Tagging with Diverse Amines Using an Engineered Sortase Variant.

Chemoenzymatic modification of proteins is an attractive option to create highly specific conjugates for therapeutics, diagnostics, or materials under gentle biological conditions. However, these methods often suffer from expensive specialized substrates, bulky fusion tags, low yields, and extra purification steps to achieve the desired conjugate. Staphylococcus aureus sortase A and its engineered variants are used to attach oligoglycine derivatives to the C-terminus of proteins expressed with a minimal LPXTG tag. This strategy has been used extensively for bioconjugation in vitro and for protein-protein conjugation in living cells. Here we show that an enzyme variant recently engineered for higher activity on oligoglycine has promiscuous activity that allows proteins to be tagged using a diverse array of small, commercially available amines, including several bioorthogonal functional groups. This technique can also be carried out in living Escherichia coli, enabling simple, inexpensive production of chemically functionalized proteins with no additional purification steps.

Controlled integration of gold nanoparticles and organic fluorophores using synthetically modified MS2 viral capsids.

The placement of fluorophores in close proximity to metal nanoparticle surfaces is proposed to enhance several photophysical properties of the dyes, potentially leading to improved quantum yields and decreased photobleaching. It is difficult in practice, however, to establish and maintain the nanoscale distances that are required to maximize these effects. The type of metal, size, and shape of the nanoparticle, the physical distance separating the metal nanoparticle from the organic dye, and the spectral properties of the fluorophore itself are all proposed to influence the quantum yield and lifetime. This results in a complex behavior that can lead to either enhanced or quenched fluorescence in different contexts. In this report, we describe a well-defined system that can be used to explore these effects, while physically preventing the fluorophores from contacting the nanoparticle surfaces. The basis of this system is the spherical protein capsid of bacteriophage MS2, which was used to house gold particles within its interior volume. The exterior surface of each capsid was then modified with Alexa Fluor 488 (AF 488) labeled DNA strands. By placing AF 488 dyes at distances of 3, 12, and 24 bp from the surface of capsids containing 10 nm gold nanoparticles, fluorescence intensity enhancements of 2.2, 1.2, and 1.0 were observed, respectively. A corresponding decrease in fluorescence lifetime was observed for each distance. Because of its well-defined and modular nature, this architecture allows the rapid exploration of the many variables involved in metal-controlled fluorescence, leading to a better understanding of this phenomenon.

Identifying and antagonizing the interactions between layilin and glycosylated collagens.

Layilin is a small type I transmembrane receptor thought to bridge extracellular ligands with the cytoskeleton through its intracellular interactions with the scaffolding protein talin. Recent bulk- and single-cell RNA sequencing experiments have repeatedly found layilin to be highly upregulated in key T cell sub-populations in multiple disease states, suggesting its importance to the adaptive immune response. Despite this prevalence, little is known about layilin's precise role in mediating extracellular interactions or how these interactions can be modulated in disease states. Here we take advantage of layilin's dependence on calcium ions to discover its interactions with highly glycosylated type II, IV, V, and VI collagens. Toward exploring layilin's role in disease, we exploited the Ca2+ dependence in a differential phage display strategy to engineer species cross-reactive antibodies that block this interaction.

Cell-surface tethered promiscuous biotinylators enable comparative small-scale surface proteomic analysis of human extracellular vesicles and cells.

Characterization of cell surface proteome differences between cancer and healthy cells is a valuable approach for the identification of novel diagnostic and therapeutic targets. However, selective sampling of surface proteins for proteomics requires large samples (>10e6 cells) and long labeling times. These limitations preclude analysis of material-limited biological samples or the capture of rapid surface proteomic changes. Here, we present two labeling approaches to tether exogenous peroxidases (APEX2 and HRP) directly to cells, enabling rapid, small-scale cell surface biotinylation without the need to engineer cells. We used a novel lipidated DNA-tethered APEX2 (DNA-APEX2), which upon addition to cells promoted cell agnostic membrane-proximal labeling. Alternatively, we employed horseradish peroxidase (HRP) fused to the glycan-binding domain of wheat germ agglutinin (WGA-HRP). This approach yielded a rapid and commercially inexpensive means to directly label cells containing common N-Acetylglucosamine (GlcNAc) and sialic acid glycans on their surface. The facile WGA-HRP method permitted high surface coverage of cellular samples and enabled the first comparative surface proteome characterization of cells and cell-derived small extracellular vesicles (EVs), leading to the robust quantification of 953 cell and EV surface annotated proteins. We identified a newly recognized subset of EV-enriched markers, as well as proteins that are uniquely upregulated on Myc oncogene-transformed prostate cancer EVs. These two cell-tethered enzyme surface biotinylation approaches are highly advantageous for rapidly and directly labeling surface proteins across a range of material-limited sample types.

Engineering luminescent biosensors for point-of-care SARS-CoV-2 antibody detection.

Current serology tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies mainly take the form of enzyme-linked immunosorbent assays, chemiluminescent microparticle immunoassays or lateral flow assays, which are either laborious, expensive or lacking sufficient sensitivity and scalability. Here we present the development and validation of a rapid, low-cost, solution-based assay to detect antibodies in serum, plasma, whole blood and to a lesser extent saliva, using rationally designed split luciferase antibody biosensors. This new assay, which generates quantitative results in 30 min, substantially reduces the complexity and improves the scalability of coronavirus disease 2019 (COVID-19) antibody tests. This assay is well-suited for point-of-care, broad population testing, and applications in low-resource settings, for monitoring host humoral responses to vaccination or viral infection.

A SARS-CoV-2 serological assay to determine the presence of blocking antibodies that compete for human ACE2 binding.

As SARS-CoV-2 continues to spread around the world, there is an urgent need for new assay formats to characterize the humoral response to infection. Convalescent serum is being used for treatment and for isolation of patient-derived antibodies. However, currently there is not a simple means to estimate serum bulk neutralizing capability. Here we present an efficient competitive serological assay that can simultaneously determine an individual's seropositivity against the SARS-CoV-2 Spike protein and estimate the neutralizing capacity of anti-Spike antibodies to block interaction with the human angiotensin converting enzyme 2 (ACE2) required for viral entry. In this ELISA-based assay, we present natively-folded viral Spike protein receptor binding domain (RBD)-containing antigens via avidin-biotin interactions. Sera are then supplemented with soluble ACE2-Fc to compete for RBD-binding serum antibodies, and antibody binding quantified. Comparison of signal from untreated serum and ACE2-Fc-treated serum reveals the presence of antibodies that compete with ACE2 for RBD binding, as evidenced by loss of signal with ACE2-Fc treatment. In our test cohort of nine convalescent SARS-CoV-2 patients, we found all patients had developed anti-RBD antibodies targeting the epitope responsible for ACE2 engagement. This assay provides a simple and high-throughput method to screen patient sera for potentially neutralizing anti-Spike antibodies to enable identification of candidate sera for therapeutic use.

Competitive SARS-CoV-2 Serology Reveals Most Antibodies Targeting the Spike Receptor-Binding Domain Compete for ACE2 Binding.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread around the world, there is an urgent need for new assay formats to characterize the humoral response to infection. Here, we present an efficient, competitive serological assay that can simultaneously determine an individual's seroreactivity against the SARS-CoV-2 Spike protein and determine the proportion of anti-Spike antibodies that block interaction with the human angiotensin-converting enzyme 2 (ACE2) required for viral entry. In this approach based on the use of enzyme-linked immunosorbent assays (ELISA), we present natively folded viral Spike protein receptor-binding domain (RBD)-containing antigens via avidin-biotin interactions. Sera are then competed with soluble ACE2-Fc, or with a higher-affinity variant thereof, to determine the proportion of ACE2 blocking anti-RBD antibodies. Assessment of sera from 144 SARS-CoV-2 patients ultimately revealed that a remarkably consistent and high proportion of antibodies in the anti-RBD pool targeted the epitope responsible for ACE2 engagement (83% ± 11%; 50% to 107% signal inhibition in our largest cohort), further underscoring the importance of tailoring vaccines to promote the development of such antibodies.IMPORTANCE With the emergence and continued spread of the SARS-CoV-2 virus, and of the associated disease, coronavirus disease 2019 (COVID-19), there is an urgent need for improved understanding of how the body mounts an immune response to the virus. Here, we developed a competitive SARS-CoV-2 serological assay that can simultaneously determine whether an individual has developed antibodies against the SARS-CoV-2 Spike protein receptor-binding domain (RBD) and measure the proportion of these antibodies that block interaction with the human angiotensin-converting enzyme 2 (ACE2) required for viral entry. Using this assay and 144 SARS-CoV-2 patient serum samples, we found that a majority of anti-RBD antibodies compete for ACE2 binding. These results not only highlight the need to design vaccines to generate such blocking antibodies but also demonstrate the utility of this assay to rapidly screen patient sera for potentially neutralizing antibodies.

Engineering luminescent biosensors for point-of-care SARS-CoV-2 antibody detection.

Current serology tests for SARS-CoV-2 antibodies mainly take the form of enzyme-linked immunosorbent assays or lateral flow assays, with the former being laborious and the latter being expensive and often lacking sufficient sensitivity and scalability. Here we present the development and validation of a rapid, low-cost solution-based assay to detect antibodies in serum, plasma, whole blood, and saliva, using rationally designed split luciferase antibody biosensors (spLUC). This new assay, which generates quantitative results in as short as 5 minutes, substantially reduces the complexity and improves the scalability of COVID-19 antibody tests for point-of-care and broad population testing.

Layilin augments integrin activation to promote antitumor immunity.

Tumor-infiltrating CD8+ T cells mediate antitumor immune responses. However, the mechanisms by which T cells remain poised to kill cancer cells despite expressing high levels of inhibitory receptors are unknown. Here, we report that layilin, a C-type lectin domain-containing membrane glycoprotein, is selectively expressed on highly activated, clonally expanded, but phenotypically exhausted CD8+ T cells in human melanoma. Lineage-specific deletion of layilin on murine CD8+ T cells reduced their accumulation in tumors and increased tumor growth in vivo. Congruently, gene editing of LAYN in human CD8+ T cells reduced direct tumor cell killing ex vivo. On a molecular level, layilin colocalized with integrin αLß2 (LFA-1) on T cells, and cross-linking layilin promoted the activated state of this integrin. Accordingly, LAYN deletion resulted in attenuated LFA-1-dependent cellular adhesion. Collectively, our results identify layilin as part of a molecular pathway in which exhausted or "dysfunctional" CD8+ T cells enhance cellular adhesiveness to maintain their cytotoxic potential.

Encapsulation of Negatively Charged Cargo in MS2 Viral Capsids.

Encapsulation into virus-like particles is an efficient way of loading cargo of interest for delivery applications. Here, we describe the encapsulation of proteins with tags comprising anionic amino acids or DNA and gold nanoparticles with negative surface charges inside MS2 bacteriophage capsids to obtain homogeneous nanoparticles with a diameter of 27 nm.

Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions.

Yeast surface display, a well-established technology for protein analysis and engineering, involves expressing a protein of interest as a genetic fusion to either the N- or C-terminus of the yeast Aga2p mating protein. Historically, yeast-displayed protein variants are flanked by peptide epitope tags that enable flow cytometric measurement of construct expression using fluorescent primary or secondary antibodies. Here, we built upon this technology to develop a new yeast display strategy that comprises fusion of two different proteins to Aga2p, one to the N-terminus and one to the C-terminus. This approach allows an antibody fragment, ligand, or receptor to be directly coupled to expression of a fluorescent protein readout, eliminating the need for antibody-staining of epitope tags to quantify yeast protein expression levels. We show that this system simplifies quantification of protein-protein binding interactions measured on the yeast cell surface. Moreover, we show that this system facilitates co-expression of a bioconjugation enzyme and its corresponding peptide substrate on the same Aga2p construct, enabling enzyme expression and catalytic activity to be measured on the surface of yeast.

Influence of Electrostatics on Small Molecule Flux through a Protein Nanoreactor.

Nature uses protein compartmentalization to great effect for control over enzymatic pathways, and the strategy has great promise for synthetic biology. In particular, encapsulation in nanometer-sized containers to create nanoreactors has the potential to elicit interesting, unexplored effects resulting from deviations from well-understood bulk processes. Self-assembled protein shells for encapsulation are especially desirable for their uniform structures and ease of perturbation through genetic mutation. Here, we use the MS2 capsid, a well-defined porous 27 nm protein shell, as an enzymatic nanoreactor to explore pore-structure effects on substrate and product flux during the catalyzed reaction. Our results suggest that the shell can influence the enzymatic reaction based on charge repulsion between small molecules and point mutations around the pore structure. These findings also lend support to the hypothesis that protein compartments modulate the transport of small molecules and thus influence metabolic reactions and catalysis in vitro.

Synthetic biologists spring into action at the 245th American Chemical Society National Meeting.

As the field of synthetic biology continues to define itself, it has merged concepts from many related areas of research: molecular biology, genetics, bioengineering, and chemistry. At the 2013 Spring American Chemical Society National Meeting in New Orleans, LA, this mixture was manifested in a wealth of sessions emphasizing the use of modern synthetic biological approaches to solve many of today's biggest chemical problems. As a result of the field's diverse yet pervasive nature, synthetic biology concepts were present in several of the conferences many divisions, including Biological Chemistry, Biochemical Technology, Cellulose and Renewable Materials, and several others. Here we offer a snapshot of some of the exciting research discussed in the dedicated synthetic biology sessions throughout the week.

Osmolyte-mediated encapsulation of proteins inside MS2 viral capsids.

The encapsulation of enzymes in nanometer-sized compartments has the potential to enhance and control enzymatic activity, both in vivo and in vitro. Despite this potential, there is little quantitative data on the effect of encapsulation in a well-defined compartment under varying conditions. To gain more insight into these effects, we have characterized two improved methods for the encapsulation of heterologous molecules inside bacteriophage MS2 viral capsids. First, attaching DNA oligomers to a molecule of interest and incubating it with MS2 coat protein dimers yielded reassembled capsids that packaged the tagged molecules. The addition of a protein-stabilizing osmolyte, trimethylamine-N-oxide, significantly increased the yields of reassembly. Second, we found that expressed proteins with genetically encoded negatively charged peptide tags could also induce capsid reassembly, resulting in high yields of reassembled capsids containing the protein. This second method was used to encapsulate alkaline phosphatase tagged with a 16 amino acid peptide. The purified encapsulated enzyme was found to have the same K(m) value and a slightly lower k(cat) value than the free enzyme, indicating that this method of encapsulation had a minimal effect on enzyme kinetics. This method provides a practical and potentially scalable way of studying the complex effects of encapsulating enzymes in protein-based compartments.