Programmable bacterial adhesion enables applications in biocatalysis, living materials, and targeted drug delivery. This functionality can be achieved through the expression of synthetic adhesins in bacterial cells. Thes...Programmable bacterial adhesion enables applications in biocatalysis, living materials, and targeted drug delivery. This functionality can be achieved through the expression of synthetic adhesins in bacterial cells. These molecules are hybrid proteins composed of four key elements: (i) a signal peptide for secretion; (ii) the beta barrel domain of an autotransporter for surface display; (iii) a nanobody module providing specific recognition and targeting capabilities; and (iv) an epitope tag for expression tracking and localization. This chapter describes a detailed protocol for visualizing these synthetic adhesins on Escherichia coli and Pseudomonas putida using indirect immunofluorescence combined with Super-Resolution Microscopy (SRM) techniques, such as STimulated Emission Depletion (STED). The workflow includes plasmid introduction into the bacterial cells, induction, immunostaining, and image acquisition to spatially track the adhesins on the cell surface.
mRNA display enables the selection of peptide and protein ligands from libraries containing more than a trillion unique sequences. Identifying optimal target binders, however, requires extensive post-selection analysis a...mRNA display enables the selection of peptide and protein ligands from libraries containing more than a trillion unique sequences. Identifying optimal target binders, however, requires extensive post-selection analysis and optimization, which is often more challenging than the selection itself. Here, we outline general strategies for analyzing mRNA display selections with the goal of advancing a small set of high-quality ligands for further study. We describe methods for sequencing and analyzing selection pools to identify potential binders, followed by experimental approaches to validate target binding and characterize ligand-target interactions. We present strategies for constructing second-generation libraries to improve initial hits, along with more specialized selection techniques for further improving binding affinity and/or protease resistance. Together, these methods provide a comprehensive strategy for post-selection analysis and the development of optimized ligands for downstream applications.
Tandem mass spectrometry (MS/MS) has become a cornerstone technology in large-scale proteomics, producing vast datasets that present considerable computational challenges for peptide identification. Conventional database...Tandem mass spectrometry (MS/MS) has become a cornerstone technology in large-scale proteomics, producing vast datasets that present considerable computational challenges for peptide identification. Conventional database-search-based approaches often exhibit limited efficiency, particularly as protein sequence repositories expand rapidly and experimental spectra grow increasingly complex. High-performance computing (HPC) offers a viable solution by supporting large-scale parallelization and algorithmic optimization. In this study, we introduce SW-Tandem, an efficient framework for peptide identification that implements parallelization of the spectrum dot product scoring algorithm on the Sunway TaihuLight supercomputer. Through the incorporation of memory-access optimization, double buffering, and vectorization techniques, SW-Tandem achieves marked improvements in computational performance while maintaining identification accuracy. Experimental evaluations reveal that SW-Tandem substantially accelerates peptide identification and surpasses existing state-of-the-art methods when deployed across multiple nodes. These findings underscore the potential of integrating HPC with algorithmic enhancements to advance large-scale bioinformatics research.
Disulfide bonds are key structural components ensuring proper protein folding. Those bonds, however, do not form in the reducing intracellular environment, often resulting in misfolded, unstable proteins with subpar func...Disulfide bonds are key structural components ensuring proper protein folding. Those bonds, however, do not form in the reducing intracellular environment, often resulting in misfolded, unstable proteins with subpar function. To enable intracellular functionality of such proteins, disulfide-free variants with restored stability and function should be engineered. Here, we present a method to substitute the cysteine residues with optimal amino acid combinations using site saturation mutagenesis and screening for improved disulfide-free variants using yeast surface display. We provide a detailed protocol to directly screen for thermal stability of proteins using conformationally specific ligands, and an alternative to harness the correlation between protein stability and expression levels in yeast. We also discuss different strategies for randomizing cysteine residues and approaches for handling proteins with multiple disulfide bonds.
Proteins are central to nearly all biological processes, mediating enzymatic catalysis, structural scaffolding, and molecular signaling. Deciphering how linear amino acid sequences encode three-dimensional conformations...Proteins are central to nearly all biological processes, mediating enzymatic catalysis, structural scaffolding, and molecular signaling. Deciphering how linear amino acid sequences encode three-dimensional conformations and biological functions has long been a defining challenge in molecular life sciences. Traditional structural biology approaches, such as X-ray crystallography and cryo-electron microscopy, have established the field, yet their high cost, labor intensity, and limited throughput restrict comprehensive coverage of the proteome. The exponential expansion of protein sequence databases, paired with advances in artificial intelligence (AI) and deep learning, has drastically accelerated our ability to predict, annotate, and even design proteins. In this mini review, we trace the evolution of AI-driven methods in protein research, from early residue-contact prediction using coevolutionary information to transformative breakthroughs, the rise of protein language models (PLMs), and the emerging era of generative design and functional modeling. Throughout, we highlight key conceptual advances and their translational implications for biomedical science and biotechnology.
mRNA display is a powerful in vitro selection technique that enables the discovery of peptide and protein ligands from libraries exceeding 10 trillion unique sequences. The technique allows for exquisite control over bin...mRNA display is a powerful in vitro selection technique that enables the discovery of peptide and protein ligands from libraries exceeding 10 trillion unique sequences. The technique allows for exquisite control over binding stringency and specificity, and it is highly amenable to high-throughput ligand profiling and integration with machine learning approaches. Here, we describe general methods for performing mRNA display selections.
Prenylated peptides, endowed with hydrophobic prenyl groups, are widely found as secondary metabolites in nature. To develop de novo artificial prenylated peptide ligands with selective affinity for a target protein of c...Prenylated peptides, endowed with hydrophobic prenyl groups, are widely found as secondary metabolites in nature. To develop de novo artificial prenylated peptide ligands with selective affinity for a target protein of choice, a random prenylated peptide library can be constructed through the combination of a custom-made in vitro translation system and a peptide prenyltransferase, which is subsequently applied to mRNA display screening. In this chapter, we describe protocols for constructing a prenylated thioether-closed macrocyclic peptide library and conducting mRNA display screening to identify selective ligands.
The phage display technology allows to select peptides, exposed on the surface of bacteriophage virions, which can specifically bind to virtually any materials, including proteins, lipids, carbohydrates, and other organi...The phage display technology allows to select peptides, exposed on the surface of bacteriophage virions, which can specifically bind to virtually any materials, including proteins, lipids, carbohydrates, and other organic macromolecules, as well as metals or metal oxides. In this chapter, we describe and discuss procedures for effective selection of peptides characterized with affinity to metallic or metal oxide nanoparticles. The use of a peptide library displayed on virions of the M13 bacteriophage, the most commonly used phage display system, is presented. Specific notes indicate what are crucial steps in the procedure, what should be avoided and why, and what should be done if common problems appear. The scheme of the biopanning process, the crucial step in selecting specific peptides, which bind to nanoparticles composed of metals or metal oxides, is presented and discussed.
Antibodies, central components of the adaptive immune system, possess the remarkable ability to specifically recognize and bind to foreign substances known as antigens. This recognition occurs through the interaction of...Antibodies, central components of the adaptive immune system, possess the remarkable ability to specifically recognize and bind to foreign substances known as antigens. This recognition occurs through the interaction of the antibody's antigen-binding site, or paratope, with a specific region on the antigen called the epitope. Understanding the precise nature of these interactions, particularly the identification and characterization of epitopes, is of paramount importance for a wide array of biomedical applications. The mapping of antibody epitopes, the process of experimentally identifying the binding site of an antibody on its target antigen, provides critical insights into antibody function and its diverse applications. Peptide phage display provides a convenient and rapid method to identify epitopes of specific monoclonal antibodies. This protocol highlights the use of a peptide phage display library for epitope identification of a recombinant monoclonal antibody.
Yeast molecular display is a convenient tool for developing and improving recombinant proteins and assessing their functions. Molecular display using Saccharomyces cerevisiae has been applied for screening valuable bioch...Yeast molecular display is a convenient tool for developing and improving recombinant proteins and assessing their functions. Molecular display using Saccharomyces cerevisiae has been applied for screening valuable biochemical molecules, including antigens and antibodies. In this chapter, we describe the genetic construction of a yeast molecular display system for a nanobody and its evaluation using fluorescence microscopy and flow cytometry.
The excessive and inappropriate use of antibiotics has driven the proliferation of antibiotic-resistant bacterial strains, posing significant challenges to conventional antimicrobial therapies. Endowed with remarkable en...The excessive and inappropriate use of antibiotics has driven the proliferation of antibiotic-resistant bacterial strains, posing significant challenges to conventional antimicrobial therapies. Endowed with remarkable engineering versatility, the M13 bacteriophage has emerged as a versatile platform for the development of novel antimicrobial strategies. In this chapter, we focused on the major coat protein pVIII of the M13 phage, utilizing genetic engineering and chemical modification approaches to engineer pVIII mutants with tailored surface charge properties. Through comparative analysis of the antibacterial efficacy of phage variants with distinct surface charges, we systematically assessed the influence of positive and negative charge densities on bactericidal activity, aiming to advance the development of innovative antimicrobial therapies.
Generally, in vitro display technology serves as a fundamental method for performing in vitro selection by linking genotypes to phenotypes. This study introduces a rapid and simple method for preparing VHH antibody-templ...Generally, in vitro display technology serves as a fundamental method for performing in vitro selection by linking genotypes to phenotypes. This study introduces a rapid and simple method for preparing VHH antibody-template DNA conjugate suitable for the LAMP method (Loop-mediated isothermal amplification), known for its rapid and highly sensitive detection, using cDNA display, an in vitro display technology.
Yeast display platform has proven instrumental for directed evolution of diverse proteins towards altered binding specificity, affinity, or improved biophysical properties. Particularly in antibody display, this method h...Yeast display platform has proven instrumental for directed evolution of diverse proteins towards altered binding specificity, affinity, or improved biophysical properties. Particularly in antibody display, this method has delivered variants that are featured today as integral parts of approved therapeutic agents. While broadly used for discovery of binding agents, yeast display has also been applied for optimization of properties of Fc fragment. Among the bispecific antibody formats, which come in a variety of different molecular architectures, the single-chain Fc is a less explored alternative. This format offers novel opportunities for positioning of binding sites in a multispecific molecule, however, necessitates special design due to its unique features such as linker. We tested the amenability of yeast system for the efficient presentation of the single-chain IgG Fc and characterized the displayed fragments for binding of structural markers and Fc gamma receptors. Taken together, this chapter sets the base for successful use of yeast display in isolation of single-chain Fc variants with tailored ligand-binding properties.
Complementary-determining regions (CDRs) are the primary factor that determines antibody diversity, while CDR H3 plays a central role in antigen binding. Randomization of this loop within a stable framework provides a po...Complementary-determining regions (CDRs) are the primary factor that determines antibody diversity, while CDR H3 plays a central role in antigen binding. Randomization of this loop within a stable framework provides a powerful strategy for generating synthetic antibody libraries. This chapter outlines an approach that is Kunkel mutagenesis based, for constructing a monoclonal antibody library diversified at CDR H3. The method uses uracilated single-stranded DNA templates and degenerate oligonucleotides to introduce targeted variability while suppressing nonrecombinant background through strategically placed stop codons and restriction sites. The resulting repertoire is directly compatible with phage display for downstream selection. This streamlined protocol enables the generation of antibody libraries suitable for applications in affinity maturation, binder discovery, and antibody development.
Phage display is a powerful high-throughput screening technique used to screen diverse peptide libraries against therapeutically relevant protein targets. Compared to linear peptides, cyclic peptides have improved stabil...Phage display is a powerful high-throughput screening technique used to screen diverse peptide libraries against therapeutically relevant protein targets. Compared to linear peptides, cyclic peptides have improved stability and binding characteristics. There are various methods to construct cyclic peptide phage display libraries, which can be screened against targets to identify cyclic peptide candidates. These peptides can then be optimized to enhance potency and specificity. In this chapter, we outline a strategy to generate cyclic peptide phage display libraries using a bischlooroxime crosslinker.
Phage display technology has emerged as an indispensable and highly successful platform for the in vitro generation of monoclonal antibodies (mAbs) against a vast spectrum of targets, ranging from disease biomarkers and...Phage display technology has emerged as an indispensable and highly successful platform for the in vitro generation of monoclonal antibodies (mAbs) against a vast spectrum of targets, ranging from disease biomarkers and native biologically important proteins to small molecules. The critical determinant for constructing a high-quality library capable of yielding high-affinity antibodies is the diversity of the source antibody genes. Naïve antibody libraries, the most common type, are typically derived from the IgM repertoire of healthy human donors, using B-cells isolated from peripheral blood mononuclear cells (PBMCs). The preferred format for these displayed antibodies is the single-chain fragment variable (scFv) due to its smaller size and functional suitability for the phage display system. This chapter details the technical workflow for harnessing this power, which involves a robust two-step cloning method for constructing the high-diversity library, followed by the iterative process of biopanning for the efficient selection and isolation of target-specific, high-affinity clones.
There are several epigenetic reader proteins that recognize butyrylated lysine residues, yet the majority peptide probes have been limited to histone-based substrates. Development of selective peptide substrates will pla...There are several epigenetic reader proteins that recognize butyrylated lysine residues, yet the majority peptide probes have been limited to histone-based substrates. Development of selective peptide substrates will play a key role in studying these proteins and in developing inhibitors of therapeutic potential. We have previously reported a strategy to incorporate N-butyryl-L-lysine into phage-displayed peptide libraries that was used to identify inhibitors for the ENL YEATS domain. Here, we describe the production of phage-displayed peptide libraries that contain butyrylated lysine residues and their subsequent use to direct phage selections toward the active sites of epigenetic proteins. We envision the technique can be applicable to performing phage selections toward many different epigenetic readers, writers, and erasers implicated in a variety of cellular processes and diseases.
Gene fusions and their associated chimeric RNAs have historically been recognized for their roles in cancer. However, recent research has increasingly detected chimeric transcripts in normal tissues and noncancerous cell...Gene fusions and their associated chimeric RNAs have historically been recognized for their roles in cancer. However, recent research has increasingly detected chimeric transcripts in normal tissues and noncancerous cell lines. Extensive efforts have begun to annotate genomic structural variation, revealing gene fusions capable of generating chimeric transcripts even in normal tissues. In this study, we present a bottom-up approach targeting population-specific chimeric RNAs, identifying 58 such instances in the GTEx cohort. Among these, notable examples include SUZ12P1-CRLF3, TFG-ADGRG7, and TRPM4-PPFIA3, each associated with ancestry. We present direct genomic evidence for 29 polymorphic chimeric RNAs associated with structural variants, including 13 previously unreported rare variants. Additionally, utilizing data from the All of Us research program alongside a substantial clinical cohort, we characterized the clinical relevance of the SUZ12P1-CRLF3-associated variant. Collectively, our findings highlight the effectiveness of leveraging population-specific chimeric transcripts, exemplified by SUZ12P1-CRLF3, as a method for uncovering otherwise elusive transcribed genomic structural variants.
Gene fusions were thought to be unique features of neoplasia. However, more and more studies have identified fusion RNAs in normal physiology. Through RNA sequencing of human normal tissues from GTEx project, many chimer...Gene fusions were thought to be unique features of neoplasia. However, more and more studies have identified fusion RNAs in normal physiology. Through RNA sequencing of human normal tissues from GTEx project, many chimeric RNAs were found. By analyzing the fusion transcriptome, we observed a close cluster between samples of the same or similar tissues, supporting the feasibility of using fusion RNA profiling to reveal connections between biological samples. To put the concept into use, we selected alveolar rhabdomyosarcoma (ARMS), a myogenic pediatric cancer whose exact cell of origin is not clear. PAX3-FOXO1 fusion RNA, which is considered a hallmark of ARMS, was found during normal muscle cell differentiation. We analyzed RNA sequencing from various time points during myogenesis and uncovered many chimeric fusion RNAs.
Recent studies have highlighted that many diseases and conditions, including aging, cancer, and infectious diseases, can manifest differently in males and females. Over the past years, sex-specific transcriptomes have be...Recent studies have highlighted that many diseases and conditions, including aging, cancer, and infectious diseases, can manifest differently in males and females. Over the past years, sex-specific transcriptomes have been uncovered in stem cells, nervous system, and immune system. However, chimeric RNAs formed by intergenic splicing are less appreciated, but provide another critical layer of RNA diversification. Leveraging the analysis of RNA-Seq data and the validation of outcomes across a cohort of over 1200 blood samples, we discovered UBA1-CDK16, a female-specific chimeric transcript. In contrast, both parental genes exhibit expression in males and females. Mechanistically, UBA1-CDK16 arises through cis-splicing between two adjacent X-linked genes, originating from the inactive X chromosome. Functionally, our investigation reveals that UBA1-CDK16 is enriched in the myeloid lineage and plays a regulatory role in myeloid differentiation. Notably, female COVID-19 patients who tested negative for this chimeric transcript displayed higher counts of neutrophils, highlighting its potential role in disease pathogenesis. These findings support the notion that chimeric RNAs represent a new repertoire of transcripts that can be regulated independently from the parental genes, and a new class of RNA variance with potential implications in sexual dimorphism and immune responses.