In the pursuit of understanding cell signaling and synthetic biology, an ability to understand and characterize phosphorylation mechanisms is indispensable. Brain-gut-microbiota axis Current techniques for characterizing kinase-substrate interactions are hampered by low throughput and the diversity of the samples under investigation. The latest advancements in yeast surface display technology present opportunities for exploring individual kinase-substrate interactions without requiring a stimulus. This document describes techniques for constructing substrate libraries within full-length domains of interest, with the intracellular co-localization of specific kinases resulting in the display of phosphorylated domains on the yeast cell surface. Enrichment strategies for these libraries based on their phosphorylation state, including fluorescence-activated cell sorting and magnetic bead selection, are further detailed.
The diverse conformations that some therapeutic targets' binding pockets can assume are, to some extent, determined by the protein's motion and its relationships with other molecules. The binding pocket's inaccessibility presents a considerable, perhaps insurmountable, obstacle to the innovative identification or optimization of small-molecule ligands. A protocol for the creation of a target protein and a yeast display FACS sorting technique is detailed here. The strategy is to identify protein variants capable of enhanced binding to a cryptic site-specific ligand, a characteristic rooted in the presence of a stable transient binding pocket. Ligand screening is made possible by the protein variants developed through this strategy, which exhibit accessible binding sites, thus potentially accelerating drug discovery.
In recent times, significant strides have been made in the development of bispecific antibodies (bsAbs), leading to a considerable collection of these therapies now being evaluated in clinical trials. In the realm of molecular design, immunoligands, multifaceted molecules, have been developed, alongside antibody scaffolds. These molecular entities typically feature a natural ligand for receptor engagement, the antibody-derived paratope enabling engagement with an additional antigen. Conditional activation of immune cells, particularly natural killer (NK) cells, is achievable using immunoliagands in response to the presence of tumor cells, leading to target-dependent tumor cell lysis. Nonetheless, a large number of naturally occurring ligands possess only a moderate affinity for their partner receptor, which may restrict the killing power of immunoligands. This document outlines protocols for affinity maturation of B7-H6, the natural ligand for NK cell-activating receptor NKp30, employing yeast surface display.
The creation of classical yeast surface display (YSD) antibody immune libraries involves the separate amplification of heavy-chain (VH) and light-chain (VL) antibody variable regions, followed by random recombination during molecular cloning. Despite the overall similarity, every B cell receptor displays a unique combination of VH and VL, chosen and refined through in vivo affinity maturation for optimal stability and antigen binding. In this way, the natural coupling of variable components within the antibody chain is key to the functioning of the antibody and its related physical attributes. The amplification of cognate VH-VL sequences is facilitated by a method compatible with both next-generation sequencing (NGS) and YSD library cloning approaches. Single B cell encapsulation within water-in-oil droplets is combined with a one-pot reverse transcription overlap extension PCR (RT-OE-PCR) for the rapid generation of a paired VH-VL repertoire from more than one million B cells in a single workday.
The immune cell profiling power of single-cell RNA sequencing (scRNA-seq) can be effectively utilized in the strategic development of theranostic monoclonal antibodies (mAbs). To establish a design framework, this method utilizes scRNA-seq to identify natively paired B-cell receptor (BCR) sequences from immunized mice, leading to a streamlined workflow for expressing single-chain antibody fragments (scFabs) on the surface of yeast, enabling high-throughput characterization and subsequent refinement via directed evolution experiments. Though this chapter isn't overly specific, this approach easily incorporates the increasing number of in silico tools designed to enhance affinity and stability, and other critical developability characteristics, like solubility and immunogenicity.
Streamlined discovery of novel antibody binders is facilitated by the use of in vitro antibody display libraries, which have emerged as powerful tools. The in vivo selection process for antibody repertoires leads to the precise pairing of variable heavy and light chains (VH and VL) with high specificity and affinity; this pairing is not preserved during the construction of in vitro recombinant libraries. A cloning method is detailed here, merging the advantages of in vitro antibody display's adaptability and diversity with those of natively paired VH-VL antibodies. In this vein, VH-VL amplicon cloning is undertaken using a two-step Golden Gate cloning method, thus permitting the display of Fab fragments on yeast cells.
Fcab fragments, which incorporate a novel antigen-binding site generated by mutating the C-terminal loops of the CH3 domain, serve as components of symmetrical, bispecific IgG-like antibodies by replacing the wild-type Fc. These proteins' homodimeric structure is usually responsible for their capacity to bind two antigen molecules. Monovalent engagement is particularly desirable in biological systems, either to prevent the adverse effects of agonistic activity and potential safety hazards, or for the appealing option of combining a single chain (namely, one half) of an Fcab fragment that binds different antigens within a single antibody. The construction and selection of yeast libraries displaying heterodimeric Fcab fragments are described, along with the effects of varying the thermostability of the underlying Fc scaffold and innovative library designs that facilitate the isolation of highly affine antigen-binding clones.
The antibody repertoire of cattle includes antibodies with remarkably long CDR3H regions, contributing to the formation of extensive knobs on their cysteine-rich stalk structures. The compact knob domain's presence enables the identification of potential antibody targets, epitopes not readily accessible to traditional antibodies. Utilizing yeast surface display and fluorescence-activated cell sorting, a high-throughput method is described for the effective access of the potential of bovine-derived antigen-specific ultra-long CDR3 antibodies, offering a straightforward approach.
Generating affibody molecules using bacterial display platforms on Gram-negative Escherichia coli and Gram-positive Staphylococcus carnosus are the subject of this review, which also explains the underlying principles. Therapeutic, diagnostic, and biotechnological avenues have recognized the potential of affibody molecules, which represent a compact and robust alternative protein scaffold. With high modularity of functional domains, they consistently manifest high levels of stability, affinity, and specificity. Small scaffold size of affibody molecules results in rapid excretion through renal filtration, making for efficient extravasation into and penetration of tissues. Both preclinical and clinical research demonstrates the safety and potential of affibody molecules as a complement to antibodies for the purposes of in vivo diagnostic imaging and therapy. Bacteria-displayed affibody libraries sorted via fluorescence-activated cell sorting represent a straightforward and effective methodology to produce novel affibody molecules with high affinity for diverse molecular targets.
In vitro phage display, a technique in antibody research, has effectively resulted in the discovery of both camelid VHH and shark VNAR variable antigen receptor domains. Exceptional length characterizes the CDRH3 in bovines, with a conserved structural pattern, encompassing a knob domain and a stalk. Antibody fragments smaller than VHH and VNAR are typically produced when the ultralong CDRH3 or the knob domain alone is removed from the antibody scaffold, enabling the fragments to bind antigens. free open access medical education Through the extraction of immune material from bovine animals and the selective amplification of knob domain DNA sequences using polymerase chain reaction, knob domain sequences are cloned into a phagemid vector, ultimately producing knob domain phage libraries. By panning libraries against a particular antigen, target-specific knob domains can be concentrated. The phage display of knob domains leverages the connection between phage genetic makeup and observable characteristics, potentially serving as a high-throughput approach to identify target-specific knob domains, thereby facilitating the exploration of the pharmacological properties inherent to this unique antibody fragment.
Therapeutic antibodies, bispecific antibodies, and chimeric antigen receptor (CAR) T-cells, in their use for cancer treatment, fundamentally utilize an antibody fragment or antibody that binds to a characteristic tumor cell surface antigen. Immunotherapy's effective antigens are, ideally, uniquely found on tumor cells or linked to them, and are expressed persistently on the tumor cell. The selection of promising proteins for optimizing immunotherapies could arise from utilizing omics methods, enabling a comparison between healthy and tumor cells, and identifying novel target structures. Despite this, the tumor cell surface's post-translational modifications and structural alterations remain difficult to identify or even impossible to access through these techniques. Mitochondrial Metabolism chemical Employing cellular screening and phage display of antibody libraries, this chapter outlines a different approach to potentially identify antibodies that target novel tumor-associated antigens (TAAs) or epitopes. The investigation into anti-tumor effector functions, facilitated by further conversion of isolated antibody fragments into chimeric IgG or other antibody formats, culminates in identifying and characterizing the corresponding antigen.
The Nobel Prize-awarded phage display technology, first appearing in the 1980s, has been a widely used technique for in vitro antibody selection, leading to discoveries in both therapeutic and diagnostic applications.