Cellular and Molecular Engineering of Glycan Sialylation in Heterologous Systems.

Glycans have been shown to play a key role in many biological processes, such as signal transduction, immunogenicity, and disease progression. Among the various glycosylation modifications found on cell surfaces and in biomolecules, sialylation is especially important, because sialic acids are typically found at the terminus of glycans and have unique negatively charged moieties associated with cellular and molecular interactions. Sialic acids are also crucial for glycosylated biopharmaceutics, where they promote stability and activity.

Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration.

The Spike protein of SARS-CoV-2, its receptor-binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus.

Recent advances in the engineering and application of streptavidin-like molecules.

Streptavidin (SA), and other related proteins, has been isolated from a wide range of organisms, including bacteria, fungi, frogs, fish, and birds. Although their original function is not well understood, they have found an important place in biotechnology based on their unique ability to bind biotin molecules with high affinity and specificity. The SA-biotin interaction is robust and easy to incorporate into different designs, and as such, it is used when reliable molecule interaction is needed under poorly controlled experimental conditions.

High-Affinity Antibody Detection with a Bivalent Circularized Peptide Containing Antibody-Binding Domains.

Direct chemical labeling of antibody produces molecules with poorly defined modifications. Use of a small antibody-binding protein as an adapter can simplify antibody functionalization by forming a specific antibody-bound complex and introducing site-specific modifications. To stabilize a noncovalent antibody complex that may be used without chemical crosslinking, a bivalent antibody-binding protein is engineered with an improved affinity of interaction by joining two Z domains with a conformationally flexible linker.

Engineered pH-dependent recycling antibodies enhance elimination of Staphylococcal enterotoxin B superantigen in mice.

A new modality in antibody engineering has emerged in which the antigen affinity is designed to be pH dependent (PHD). In particular, combining high affinity binding at neutral pH with low affinity binding at acidic pH leads to a novel antibody that can more effectively neutralize the target antigen while avoiding antibody-mediated antigen accumulation. Here, we studied how the in vivo pharmacokinetics of the superantigen, Staphylococcal enterotoxin B (SEB), is affected by an engineered antibody with pH-dependent binding.

Functional expression of monomeric streptavidin and fusion proteins in Escherichia coli: applications in flow cytometry and ELISA.

Monomeric streptavidin (mSA) offers a combination of structural and binding properties that are useful in many applications, including a small size and monovalent biotin binding. Because mSA contains a structurally important disulfide bond, the molecule does not fold correctly when expressed inside the cell. We show that mSA can be expressed in a functional form in Escherichia coli by fusing the OmpA signal sequence at the amino terminus.

Super-resolution imaging of synaptic and Extra-synaptic AMPA receptors with different-sized fluorescent probes.

Previous studies tracking AMPA receptor (AMPAR) diffusion at synapses observed a large mobile extrasynaptic AMPAR pool. Using super-resolution microscopy, we examined how fluorophore size and photostability affected AMPAR trafficking outside of, and within, post-synaptic densities (PSDs) from rats. Organic fluorescent dyes (≈4 nm), quantum dots, either small (≈10 nm diameter; sQDs) or big (>20 nm; bQDs), were coupled to AMPARs via different-sized linkers.

Enhancement of Muramyl Dipeptide-Dependent NOD2 Activity by a Self-Derived Peptide.

Nucleotide-binding and oligomerization domain like receptors (NLR) are pattern recognition receptors used to provide rapid immune response by detecting intracellular pathogen-associated molecules. Loss of NLR activity is implicated in genetic disorders, disruption of adaptive immunity, and chronic inflammation. One NLR protein, NOD2, is frequently mutated in Crohn's disease (CD), which is an inflammatory disease of the gastrointestinal tract.

Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin.

The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes.

Postsynthetic Domain Assembly with NpuDnaE and SspDnaB Split Inteins.

Inteins are protein segments embedded in frame within a precursor sequence that catalyze a self-excision reaction and ligate the flanking sequences with a standard peptide bond. Split inteins are expressed as two separate polypeptide fragments and trans-splice upon subunit association. Split inteins have found use in biotechnology applications but their use in postsynthetic domain assembly in vivo has been limited to the ligation of two protein domains.

Epitope-Specific Binder Design by Yeast Surface Display.

Yeast surface display is commonly used to engineer affinity and design novel molecular interaction. By alternating positive and negative selections, yeast display can be used to engineer binders that specifically interact with the target protein at a defined site. Epitope-specific binders can be useful as inhibitors if they bind the target molecule at functionally important sites.

Selective TERS detection and imaging through controlled plasmonics.

Enhanced Raman spectroscopy offers capabilities to detect molecules in the complex molecular environments and image chemical heterogeneity in a wide range of samples. It has been shown that plasmonic interactions between a TERS tip and a metal surface produce significant enhancements. In this report we show how SERS spectra from purified molecules can be used to selectively image proteins on surfaces and in cell membranes.

Expression and purification of soluble monomeric streptavidin in Escherichia coli.

We recently reported the engineering of monomeric streptavidin (mSA) for use in monomeric detection of biotinylated ligands. Although mSA can be expressed functionally on the surface of mammalian cells and yeast, the molecule does not fold correctly when expressed in Escherichia coli. Refolding from inclusion bodies is cumbersome and yields a limited amount of purified protein.

Streptavidin-biotin technology: improvements and innovations in chemical and biological applications.

Streptavidin and its homologs (together referred to as streptavidin) are widely used in molecular science owing to their highly selective and stable interaction with biotin. Other factors also contribute to the popularity of the streptavidin-biotin system, including the stability of the protein and various chemical and enzymatic biotinylation methods available for use with different experimental designs. The technology has enjoyed a renaissance of a sort in recent years, as new streptavidin variants are engineered to complement native proteins and novel methods of introducing selective biotinylation are developed for in vitro and in vivo applications.

Structure-based engineering of streptavidin monomer with a reduced biotin dissociation rate.

We recently reported the engineering of monomeric streptavidin, mSA, corresponding to one subunit of wild type (wt) streptavidin tetramer. The monomer was designed by homology modeling, in which the streptavidin and rhizavidin sequences were combined to engineer a high affinity binding pocket containing residues from a single subunit only. Although mSA is stable and binds biotin with nanomolar affinity, its fast off rate (koff ) creates practical challenges during applications.

Epitope-guided engineering of monobody binders for in vivo inhibition of Erk-2 signaling.

Although the affinity optimization of protein binders is straightforward, engineering epitope specificity is more challenging. Targeting a specific surface patch is important because the biological relevance of protein binders depends on how they interact with the target. They are particularly useful to test hypotheses motivated by biochemical and structural studies.

Stable, high-affinity streptavidin monomer for protein labeling and monovalent biotin detection.

The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit.

Biotin-assisted folding of streptavidin on the yeast surface.

Yeast surface display allows heterologously expressed proteins to be targeted to the exterior of the cell wall and thus has a potential as a biotechnology platform. In this study, we report the successful display of functional streptavidin on the yeast surface. Streptavidin binds the small molecule biotin with high affinity (K(d) ≈ 10(-14)M) and is used widely in applications that require stable noncovalent interaction, including immobilization of biotinylated compounds on a solid surface.

Engineered streptavidin monomer and dimer with improved stability and function.

Although streptavidin's high affinity for biotin has made it a widely used and studied binding protein and labeling tool, its tetrameric structure may interfere with some assays. A streptavidin mutant with a simpler quaternary structure would demonstrate a molecular-level understanding of its structural organization and lead to the development of a novel molecular reagent. However, modulating the tetrameric structure without disrupting biotin binding has been extremely difficult.

Computational and mutagenesis studies of the streptavidin native dimer interface.

Wt streptavidin forms a domain swapped tetramer consisting of two native dimers. The role of tetramerization has been studied previously and is known to contribute to biotin binding by allowing the exchange of W120 between adjacent subunits. However, the role of dimer formation in streptavidin folding and function has been largely overlooked to date, although native dimers are necessary for tetramer formation and thus for high affinity biotin binding.

Flow cytometric analysis of genetic FRET detectors containing variable substrate sequences.

A genetic Fluorescence Resonance Energy Transfer (FRET) detector undergoes a post-translational modification (PTM)-induced conformational change that results in increased FRET. To test if the PTM-dependent FRET change can be quantified by flow cytometry, we purified and immobilized a genetic detector on microbeads and used flow cytometry to measure its FRET efficiency before and after Erk-2-mediated phosphorylation. The fluorescence ratio R between the acceptor and donor fluorescence, which was obtained by fitting a straight line through the data points in linear space, increases following phosphorylation, thus demonstrating that flow cytometry is capable of detecting a PTM-dependent FRET response.

Disulfide trapping of protein complexes on the yeast surface.

Protein complexes are common in nature and play important roles in biology, but studying the quaternary structure formation in vitro is challenging since it involves lengthy and expensive biochemical steps. There are frequent technical difficulties as well with the sensitivity and resolution of the assays. In this regard, a technique that can analyze protein-protein interactions in high throughput would be a useful experimental tool.

Computational design of protein therapeutics.

Computation is increasingly used to guide protein therapeutic designs. Some of the potential applications for computational, structure-based protein design include antibody affinity maturation, modulation of protein-protein interaction, stability improvement and minimization of protein aggregation. The versatility of a computational approach is that different biophysical properties can be analyzed on a common framework.