Application of the surface engineered recombinant Escherichia coli to the industrial battery waste solution for lithium recovery.

Escherichia coli were engineered to selectively adsorb and recover lithium from the environment by employing a bacterial cell surface display strategy. Lithium binding peptide (LBP1) was integrated into the Escherichia coli membrane protein OmpC. The effect of environmental conditions on the adsorption of lithium by a recombinant strain was evaluated, and lithium particles on the cellular surface were analyzed by FE-SEM and XRD. To elevate the lithium adsorption, dimeric, trimeric, and tetrameric repeats of the LBP1 peptide were constructed and displayed on the surface of E. coli. The constructed recombinant E. coli displaying the LBP1 trimer was applied to real industrial lithium battery wastewater to recover lithium.

Quantitative measurement of cell-surface displayed proteins based on split-GFP assembly.

BACKGROUND: Microbial cell surface display technology allows immobilizing proteins on the cell surface by fusing them to anchoring motifs, thereby endowing the cells with diverse functionalities. However, the assessment of successful protein display and the quantification of displayed proteins remain challenging. The green fluorescent protein (GFP) can be split into two non-fluorescent fragments, while they spontaneously assemble and emit fluorescence when brought together through complementation. Based on split-GFP assembly, we aim to: (1) confirm the success display of passenger proteins, (2) quantify the number of passenger proteins displayed on individual cells. RESULTS: In this study, we propose two innovative methods based on split-green fluorescent protein (split-GFP), named GFP1-10/GFP11 and GFP1-9/GFP10-11 assembly, for the purpose of confirming successful display and quantifying the number of proteins displayed on individual cells. We evaluated the display efficiency of SUMO and ubiquitin using different anchor proteins to demonstrate the feasibility of the two split-GFP assembly systems. To measure the display efficiency of functional proteins, laccase expression was measured using the split-GFP assembly system by co-displaying GFP11 or GFP10-11 tags, respectively. CONCLUSIONS: Our study provides two split-GFP based methods that enable qualitative and quantitative analyses of individual cell display efficiency with a simple workflow, thus facilitating further comprehensive investigations into microbial cell surface display technology. Both split-GFP assembly systems offer a one-step procedure with minimal cost, simplifying the fluorescence analysis of surface-displaying cells.

Biosensing-based quality control monitoring of the higher-order structures of therapeutic antibody domains.

Advanced biopharmaceutical manufacturing requires novel process analytical technologies for the rapid and sensitive assessment of the higher-order structures of therapeutic proteins. However, conventional physicochemical analyses of denatured proteins have limitations in terms of sensitivity, throughput, analytical resolution, and real-time monitoring capacity. Although probe-based sensing can overcome these limitations, typical non-specific probes lack analytical resolution and provide little to no information regarding which parts of the protein structure have been collapsed. To meet these analytical demands, we generated biosensing probes derived from artificial proteins that could specifically recognize the higher-order structural changes in antibodies at the protein domain level. Biopanning of phage-displayed protein libraries generated artificial proteins that bound to a denatured antibody domain, but not its natively folded structure, with nanomolar affinity. The protein probes not only recognized the higher-order structural changes in intact IgGs but also distinguished between the denatured antibody domains. These domain-specific probes were used to generate response contour plots to visualize the antibody denaturation caused by various process parameters, such as pH, temperature, and holding time for acid elution and virus inactivation. These protein probes can be combined with established analytical techniques, such as surface plasmon resonance for real-time monitoring or plate-based assays for high-throughput analysis, to aid in the development of new analytical technologies for the process optimization and monitoring of antibody manufacturing.

Characteristics of cold-adapted carbonic anhydrase and efficient carbon dioxide capture based on cell surface display technology.

Carbonic anhydrase (CA) is currently under investigation because of its potential to capture CO2. A novel N-domain of ice nucleoproteins (INPN)-mediated surface display technique was developed to produce CA with low-temperature capture CO2 based on the mining and characterization of Colwellia sp. CA (CsCA) with cold-adapted enzyme structural features and catalytic properties. CsCA and INPN were effectively integrated into the outer membrane of the cell as fusion proteins. Throughout the display process, the integrity of the membrane of engineered bacteria BL21/INPN-CsCA was maintained. Notably, the study affirmed positive applicability, wherein 94 % activity persisted after 5 d at 15 °C, and 73 % of the activity was regained after 5 cycles of CO2 capture. BL21/INPN-CsCA displayed a high CO2 capture capacity of 52 mg of CaCO3/mg of whole-cell biocatalysts during CO2 mineralization at 25 °C. Therefore, the CsCA functional cell surface display technology could contribute significantly to environmentally friendly CO2 capture.

Protocols for Building and Producing High Diversity Peptide Phage Display Libraries.

Phage display is an important technology to study protein-protein interaction and protein evolution, with applications in basic science and applied biotechnology, such as drug discovery and the development of targeted therapies. However, in order to be successful during a phage display screening, it is paramount to have good phage libraries. Here, we described detailed procedures to generate peptide phage display libraries with high diversity and billions of transformants.

Generation of a Naïve Human scFv Phage Display Library and Panning Selection.

Phage display antibody libraries have been successfully used as the essential tool to produce monoclonal antibodies against a plethora of targets ranging from diseases to native biologically important proteins as well as small molecules. It is well documented that diverse antibody genes are the major genetic source for the construction of a high-quality antibody library and selection of high-affinity antibodies. Naïve antibody libraries are derived using the IgM repertoire of healthy donors obtained from B-cells isolated from human peripheral blood mononuclear cell (PBMC). Single-chain fragment variable (scFv) is a routinely used format due to its smaller size and preference for phage display. The process involves the use of a two-step cloning method for library construction. The protocol also covers the biopanning process for target positive clone selection.

Understanding the Structural Requirements of Peptide-Protein Interaction and Applications for Peptidomimetic Development.

Protein-protein interaction is at the heart of most biological processes, and small peptides that bind to protein binding sites are resourceful tools to explore and understand the structural requirements for these interactions. In that sense, phage display is a well-suited technology to study protein-protein interactions, as it allows for unbiased screening of billions of peptides in search for those that interact with a protein binding domain. Here, we will illustrate how two distinct but complementary approaches, phage display and nuclear magnetic resonance (NMR), can be utilized to unveil structural details of peptide-protein interaction. Finally, knowledge derived from phage mutagenesis and NMR studies can be streamlined for quick peptidomimetic design and synthesis using the retroinversion approach to validate using in vitro and in vivo assays the therapeutic potential of peptides identified by phage display.

Preparation of Bioconjugates of Chimeric M13 Phage and Gold Nanorods.

Phage-nanomaterial conjugates are functional bio-nanofibers with various applications. While phage display can select for phages with desired genetically encoded functions and properties, nanomaterials can endow the phages with additional features at nanoscale dimensions. Therefore, combining phages with nanotechnology can construct bioconjugates with unique characteristics. One strategy for filamentous phages is to adsorb nanoparticles onto the side wall, composed of pVIII subunits, through electrostatic interactions. However, a noncovalent approach may cause offloading if the environment changes, potentially causing side effects especially for in vivo applications. Therefore, building stable phage-bioconjugates is an important need. We previously reported the construction of chimeric M13 phage conjugated with gold nanorods, named "phanorods," without weakening the binding affinity to the bacterial host cells. Herein, we give a detailed protocol for preparing the chimeric M13 phage and covalently conjugating gold nanorods to the phage.

Exploring the short linear motif-mediated protein-protein interactions of CrkL through ProP-PD.

Adaptor proteins play a pivotal role in cellular signaling mediating a multitude of protein-protein interaction critical for cellular homeostasis. Dysregulation of these interactions has been linked to the onset of various cancer pathologies and exploited by viral pathogens during host cell takeover. CrkL is an adaptor protein composed of an N-terminal SH2 domain followed by two SH3 domains that mediate interactions with diverse partners through the recognition of specific binding motifs. In this study, we employed proteomic peptide-phage display (ProP-PD) to comprehensively explore the short linear motif (SLiM)-based interactions of CrkL. Furthermore, we scrutinized how the binding affinity for selected peptides was influenced in the context of the full-length CrkL versus the isolated N-SH3 domain. Importantly, our results provided insights into SLiM-binding sites within previously reported interactors, as well as revealing novel human and viral ligands, expanding our understanding of the interactions mediated by CrkL and highlighting the significance of SLiM-based interactions in mediating adaptor protein function, with implications for cancer and viral pathologies.

Deep mining of antibody phage-display selections using Oxford Nanopore Technologies and Dual Unique Molecular Identifiers.

Antibody phage-display technology identifies antibody-antigen interactions through multiple panning rounds, but traditional screening gives no information on enrichment or diversity throughout the process. This results in the loss of valuable binders. Next Generation Sequencing can overcome this problem. We introduce a high accuracy long-read sequencing method based on the recent Oxford Nanopore Technologies (ONT) Q20 + chemistry in combination with dual unique molecular identifiers (UMIs) and an optimized bioinformatic analysis pipeline to monitor the selections. We identified binders from two single-domain antibody libraries selected against a model protein. Traditional colony-picking was compared with our ONT-UMI method. ONT-UMI enabled monitoring of diversity and enrichment before and after each selection round. By combining phage antibody selections with ONT-UMIs, deep mining of output selections is possible. The approach provides an alternative to traditional screening, enabling diversity quantification after each selection round and rare binder recovery, even when the dominating binder was > 99% abundant. Moreover, it can give insights on binding motifs for further affinity maturation and specificity optimizations. Our results demonstrate a platform for future data guided selection strategies.

Chimeric antigens displaying GPR65 extracellular loops on a soluble scaffold enabled the discovery of antibodies, which recognized native receptor.

GPR65 is a proton-sensing G-protein coupled receptor associated with multiple immune-mediated inflammatory diseases, whose function is relatively poorly understood. With few reagents commercially available to probe the biology of receptor, generation of an anti-GPR65 monoclonal antibody was desired. Using soluble chimeric scaffolds, such as ApoE3, displaying the extracellular loops of GPR65, together with established phage display technology, native GPR65 loop-specific antibodies were identified. Phage-derived loop-binding antibodies recognized the wild-type native receptor to which they had not previously been exposed, generating confidence in the use of chimeric soluble proteins to act as efficient surrogates for membrane protein extracellular loop antigens. This technique provides promise for the rational design of chimeric antigens in facilitating the discovery of specific antibodies to GPCRs.

This technique offers a viable approach for antibody discovery to difficult GPCRs.Structurally relevant, soluble chimeric scaffold proteins of GPR65 were generated.Chimeric antigens were used to identify GPR65-specific antibodies by phage display.

Review of phage display: A jack-of-all-trades and master of most biomolecule display.

Phage display was first described by George P. Smith when it was shown that virus particles were capable of presenting foreign proteins on their surface. The technology has paved the way for the evolution of various biomolecules presentation and diverse selection strategies. This unique feature has been applied as a versatile platform for numerous applications in drug discovery, protein engineering, diagnostics, and vaccine development. Over the decades, the limits of biomolecules displayed on phage particles have expanded from peptides to proteomes and even alternative scaffolds. This has allowed phage display to be viewed as a versatile display platform to accommodate various biomolecules ranging from small peptides to larger proteomes which has significantly impacted advancements in the biomedical industry. This review will explore the vast array of biomolecules that have been successfully employed in phage display technology in biomedical research.

Isolation and characterization of a novel single-chain variable fragment (scFv) against Lymphocyte function-associated antigen-1 (LFA-1) using phage display method.

Lymphocyte function-associated antigene-1 (LFA-1) is a well-described integrin found on lymphocytes and other leukocytes, which is known to be overexpressed in leukemias and lymphomas. This receptor plays a significant role in immune responses such as T-cell activation, leukocyte cell-cell interactions, and trafficking of leukocyte populations. Subsequently, binders of LFA-1 emerge as potential candidates for cancer and autoimmune therapy. This study used the phage display technique to construct and characterize a high-affinity single-chain fragment variable (scFv) antibody against LFA-1. After expression, purification, dialysis, and concentration of the recombinant LFA-1 protein, four female BALB/c mice were immunized, splenocyte's mRNA was extracted, and cDNA was synthesized. A scFv library was constructed by linking the amplified VH/Vκ fragments through a 72-bp linker using SOEing PCR. Next, the scFv gene fragments were cloned into the pComb-3XSS phagemid vector; thus, the phage library was developed. The selection process involved three rounds of phage-bio-panning, polyclonal, and monoclonal phage ELISA. AF17 was chosen and characterized among the positive clones through SDS-PAGE, Western blotting, indirect ELISA, and in-silico analyses. The results of the study showed the successful construction of a high-affinity scFv library against LFA-1. The accuracy of the AF17 production and its ability to bind to the LFA-1 were confirmed through SDS-PAGE, Western blot, and ELISA. This study highlights the potential application of the high-affinity AF17 against LFA-1 for targeting T lymphocytes for therapeutic purposes.

Screening, Expression and Identification of Nanobody Against Monkeypox Virus A35R.

Introduction: The monkeypox (Mpox) virus epidemic presents a significant risk to global public health security. A35R, a crucial constituent of EEV, plays a pivotal role in virus transmission, serves as a vital target for vaccine development, and has potential for serological detection. Currently, there is a dearth of research on nanobodies targeting A35R. The purpose of this study is to identify specific nanobodies target A35R, so as to provide new antibody candidates for Mpox vaccine development and diagnostic kit development. Methods: Three nanobodies specific to the monkeypox virus protein A35R were screened from a naïve phage display library. After four rounds of panning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA). Further, the nanobody fusion protein was constructed in pNFCG1-IgG1-Fc vector and expressed in HEK293F cells and purified by affinity chromatography. The specificity and affinity of the nanobodies were identified by ELISA. The binding kinetics of the VHH antibody to A35R were assessed via employment of a bio-layer interferometry (BLI) apparatus, thereby determining the nanobodies affinity. Results: The three purified nanobodies showed specific high-affinity binding MPXV A35R, of them, VHH-1 had the best antigen binding affinity (EC50 = 0.010 ug/mL). In addition, VHH-1 on Protein A biosensor can bind Mpox virus A35R, with an affinity constant of 54 nM as determined in BLI assay. Conclusion: In sum, we has obtained three nanobody strains against Mpox virus A35R with significant affinity and specificity, therefore laying an essential foundation for further research as well as the applications of diagnostic and therapeutic tools of Mpox virus.

Brain-Penetrating Peptide Shuttles across the Blood-Brain Barrier and Extracellular-like Space.

Systemic delivery of therapeutics into the brain is greatly impaired by multiple biological barriers─the blood-brain barrier (BBB) and the extracellular matrix (ECM) of the extracellular space. To address this problem, we developed a combinatorial approach to identify peptides that can shuttle and transport across both barriers. A cysteine-constrained heptapeptide M13 phage display library was iteratively panned against an established BBB model for three rounds to select for peptides that can transport across the barrier. Using next-generation DNA sequencing and in silico analysis, we identified peptides that were selectively enriched from successive rounds of panning for functional validation in vitro and in vivo. Select peptide-presenting phages exhibited efficient shuttling across the in vitro BBB model. Two clones, Pep-3 and Pep-9, exhibited higher specificity and efficiency of transcytosis than controls. We confirmed that peptides Pep-3 and Pep-9 demonstrated better diffusive transport through the extracellular matrix than gold standard nona-arginine and clinically trialed angiopep-2 peptides. In in vivo studies, we demonstrated that systemically administered Pep-3 and Pep-9 peptide-presenting phages penetrate the BBB and distribute into the brain parenchyma. In addition, free peptides Pep-3 and Pep-9 achieved higher accumulation in the brain than free angiopep-2 and may exhibit brain targeting. In summary, these in vitro and in vivo studies highlight that combinatorial phage display with a designed selection strategy can identify peptides as promising carriers, which are able to overcome the multiple biological barriers of the brain and shuttle different-sized molecules from small fluorophores to large macromolecules for improved delivery into the brain.

A bivalent form of a RBD-specific synthetic antibody effectively neutralizes SARS-CoV-2 variants.

Coronavirus Disease 2019 (COVID-19) pandemic is severely impacting the world, and tremendous efforts have been made to deal with it. Despite many advances in vaccines and therapeutics, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains an intractable challenge. We present a bivalent Receptor Binding Domain (RBD)-specific synthetic antibody, specific for the RBD of wild-type (lineage A), developed from a non-antibody protein scaffold composed of LRR (Leucine-rich repeat) modules through phage display. We further reinforced the unique feature of the synthetic antibody by constructing a tandem dimeric form. The resulting bivalent form showed a broader neutralizing activity against the variants. The in vivo neutralizing efficacy of the bivalent synthetic antibody was confirmed using a human ACE2-expressing mouse model that significantly alleviated viral titer and lung infection. The present approach can be used to develop a synthetic antibody showing a broader neutralizing activity against a multitude of SARS-CoV-2 variants.

Single domain antibodies specific for HER2 dimerization domain effectively disrupts HER2 dimerization.

Dimer-dependent phosphorylation of HER2 receptor is a key event for the signal transduction of HER family of receptors which correlates with tumor invasion and metastasis. New generation of therapies based on dimerization domain inhibition using monoclonal or fragment antibodies was introduced. A potent method for manufacturing antibodies and antibody fragments is the phage display antibody library method. A recombinant phage was generated using the phage display method from synthetic dAb library. Subtractive biopanning was performed on sepharose 4b resin. Evaluation of success of subtractive biopanning was confirmed by the PCR fingerprinting after the fourth round of biopanning. The fourth round of biopanning results in the isolation of several dimerization domain reactive clones based on the polyclonal phage ELISA results. Monoclonal phage cell ELISA was used to select the positive clones with the highest affinity, and they were subsequently employed for functional tests. Cell-ELISA, MTT assay and dimerization inhibition test revealed that the reactivity and specificity of the selected monoclonal phage to dimerization domain of HER2. Further, Annexin V/PI staining and gene expression analysis showed that increased apoptosis rates. Also, in silico binding of the selected clones to conformational structure of HER2 was applied, using protein-protein docking tool of the ICM-Pro software, and showed sdAbs were specifically interacted with dimerization domain of the receptor. In conclusion, we have identified a single domain targeting HER2 dimerization, which represents a promising therapeutic and diagnostic candidate for HER2-positive cancers. Purified sdAb needs to more research to evaluate it both in vivo and in vitro via functional tests to determine if it can be applied for treatment and diagnostics.

Massively-multiplexed epitope mapping techniques for viral antigen discovery.

Following viral infection, viral antigens bind specifically to receptors on the surface of lymphocytes thereby activating adaptive immunity in the host. An epitope, the smallest structural and functional unit of an antigen, binds specifically to an antibody or antigen receptor, to serve as key sites for the activation of adaptive immunity. The complexity and diverse range of epitopes are essential to study and map for the diagnosis of disease, the design of vaccines and for immunotherapy. Mapping the location of these specific epitopes has become a hot topic in immunology and immune therapy. Recently, epitope mapping techniques have evolved to become multiplexed, with the advent of high-throughput sequencing and techniques such as bacteriophage-display libraries and deep mutational scanning. Here, we briefly introduce the principles, advantages, and disadvantages of the latest epitope mapping techniques with examples for viral antigen discovery.

Construction of Human Immune and Naive scFv Phage Display Libraries.

Antibody phage display is a widely used in vitro selection technology for the generation of human recombinant antibodies and has yielded thousands of useful antibodies for research, diagnostics, and therapy. In order to successfully generate antibodies using phage display, the basis is the construction of high-quality antibody gene libraries. Here, we describe detailed methods for the construction of such high-quality immune and naive scFv gene libraries of human origin. These protocols were used to develop human naive (e.g., HAL9/10) and immune libraries, which resulted in thousands of specific antibodies for all kinds of applications.

Antibody Phage Display.

The application of antibodies has transcended across many areas of work but mainly as a research tool, for diagnostic and for therapeutic applications. Antibodies are immunoproteins from vertebrates that have the unique property of specifically binding foreign molecules and distinguish target antigens. This property allows antibodies to effectively protect the host from infections. Apart from the hybridoma technology using transgenic animals, antibody phage display is commonly considered the gold standard technique for the isolation of human monoclonal antibodies. The concept of antibody phage display surrounds the ability to display antibody fragments on the surface of M13 bacteriophage particles with the corresponding gene packaged within the particle. A repetitive in vitro affinity based selection process permits the enrichment of target specific binders. This process of recombinant human monoclonal antibody generation also enables additional engineering for various applications. This makes phage display an indispensable technique for antibody development and engineering activities.