Functional decoration of elastin-like polypeptides-based nanoparticles with a modular assembly via isopeptide bond formation.

Temperature-responsive elastin-like polypeptides (ELPs) exhibit a low critical solution temperature-type phase transition and offer potential as useful materials for the construction of nanoparticles. Herein, we developed a novel decoration method for ELP-based nanoparticles via isopeptide bond formation with the SnoopTag/SnoopCatcher system that is not affected by the heating process required for particle formation. A mixture of a fusion protein of ELP and poly(aspartic acid) (poly(D)), known as ELP-poly(D), and ELP-poly(D) fused with SnoopCatcher (ELP-poly(D)-SnC) formed protein nanoparticles as a result of the temperature responsiveness of ELP, with the resultant nanoparticles displaying the SnoopCatcher binding domain on their surfaces.

Cholesterol- and ssDNA-binding fusion protein-mediated DNA tethering on the plasma membrane.

DNA modification of the plasma membrane is an excellent approach for controlling membrane-protein interactions, modulating cell-cell/cell-biomolecule interactions, and extending the biosensing field. The hydrophobic insertion of DNA conjugated with hydrophobic anchoring molecules is utilized for tethering DNA on the cell membrane. In this study, we developed an alternative approach to tether DNA on the plasma membrane based on ssDNA- and cholesterol-binding proteins.

Sensitive Detection of Tumor Cells Using Protein Nanoparticles with Multiple Displays of DNA Aptamers and Bioluminescent Reporters.

Simple and effective detection methods for circulating tumor cells are essential for early detection and progression monitoring of tumors. The use of DNA aptamer and bioluminescence is expected to be a key tool for the simple, effective, and sensitive detection of tumor cells. Herein, we designed multifunctional protein nanoparticles for the detection of tumor cells using DNA aptamer and bioluminescence.

Development of an enhanced immunoassay based on protein nanoparticles displaying an IgG-binding domain and luciferase.

Detection of small amounts of target molecules with high sensitivity is important for the diagnosis of many diseases, including cancers, and is particularly important to detect early stages of disease. Here, we report the development of a temperature-responsive fusion protein (ELP-DCN) comprised of an elastin-like polypeptide (ELP), poly-aspartic acid (D), antibody-binding domain C (C), and NanoLuc luciferase (N). ELP-DCN proteins form nanoparticles above a certain threshold temperature that display an antibody-binding domain and NanoLuc luciferase on their surface.

Antibody response to the first dose of AZD1222 vaccine in COVID-19 convalescent and uninfected individuals in Bangladesh.

Background: Vaccination with the Oxford-AstraZeneca COVID-19 vaccine (AZD1222) initially started in the UK and quickly implemented around the Globe, including Bangladesh. Up to date, more than nine million doses administrated to the Bangladeshi public.

Method: Herein, we studied the antibody response to the first dose of AZD1222 in 86 Bangladeshi individuals using in-house ELISA kits.

Detection of SARS-CoV-2 by antigen ELISA test is highly swayed by viral load and sample storage condition.

Background: Rapid increase in COVID-19 suspected cases has rendered disease diagnosis challenging, mainly depending upon RT-qPCR. Reliable, rapid, and cost-effective diagnostic assays that complement RT-qPCR should be introduced after thoroughly evaluating their performance upon various disease phases, viral load, and sample storage conditions.

Objective: We investigated the correlation of cycle threshold (Ct) value, which implies the viral load and infection phase, and the storage condition of the clinical specimen with the diagnosis of SARS-CoV-2 through our newly developed in-house rapid enzyme-linked immunosorbent assay (ELISA) system.

AuNP Coupled Rapid Flow-Through Dot-Blot Immuno-Assay for Enhanced Detection of SARS-CoV-2 Specific Nucleocapsid and Receptor Binding Domain IgG.

Background: Serological tests detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are widely used in seroprevalence studies and evaluating the efficacy of the vaccination program. Some of the widely used serological testing techniques are enzyme-linked immune-sorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and lateral flow immunoassay (LFIA). However, these tests are plagued with low sensitivity or specificity, time-consuming, labor-intensive, and expensive.

Longitudinal Antibody Dynamics Against Structural Proteins of SARS-CoV-2 in Three COVID-19 Patients Shows Concurrent Development of IgA, IgM, and IgG.

Background: Dynamics and persistence of neutralizing and non-neutralizing antibodies can give us the knowledge required for serodiagnosis, disease management, and successful vaccine design and development. The disappearance of antibodies, absence of humoral immunity activation, and sporadic reinfection cases emphasize the importance of longitudinal antibody dynamics against variable structural antigens.

Methods: In this study, twenty-five healthy subjects working in a SARS-COV-2 serodiagnostic assay development project were enrolled, and their sign and symptoms were followed up to six months.

Development and performance evaluation of a rapid in-house ELISA for retrospective serosurveillance of SARS-CoV-2.

Background: In the ongoing pandemic situation of COVID-19, serological tests can complement the molecular diagnostic methods, and can be one of the important tools of sero-surveillance and vaccine evaluation.

Aim: To develop and evaluate a rapid SARS-CoV-2 specific ELISA for detection of anti-SARS-CoV2 IgG from patients' biological samples.

Methods: In order to develop this ELISA, three panels of samples (n = 184) have been used: panel 1 (n = 19) and panel 2 (n = 60) were collected from RT-PCR positive patients within 14 and after 14 days of onset of clinical symptoms, respectively; whereas panel 3 consisted of negative samples (n = 105) collected either from healthy donors or pre-pandemic dengue patients.

Construction of DNA-displaying nanoparticles by enzymatic conjugation of DNA and elastin-like polypeptides using a replication initiation protein.

DNA-displaying nanoparticles comprised of conjugates of single-stranded DNA (ssDNA) and elastin-like polypeptide (ELP) were developed. ssDNA was enzymatically conjugated to ELPs via a catalytic domain of Porcine Circovirus type 2 replication initiation protein (pRep) fused to ELPs. Nanoparticles were formed upon heating to temperatures above the phase transition temperature due to the hydrophobicity of ELPs and the hydrophilicity of conjugated ssDNA.

Construction of multifunctional fusion proteins with a laminin-derived short peptide to promote neural differentiation of mouse induced pluripotent stem cells.

There is growing interest in the functional roles of the extracellular matrix (ECM) in regulating the fate of pluripotent stem cells (PSCs). An artificially bioengineered ECM provides an excellent model for studying the molecular mechanisms underlying self-renewal and differentiation of PSCs, without multiple unknown and variable factors associated with natural substrates. Here, we have engineered multifunctional fusion proteins that are based on peptides from laminin, including p20, RGD, and elastin-like polypeptide (ELP), where laminin peptides work as cell adhesion molecules (CAMs) and ELP to promote anchorage.

Direct Labeling of Protein Nanoparticles with Fluorescent Compounds for Immunoassay Applications.

A fusion protein, designated ELP-D-C, comprised of a hydrophobic elastin-like polypeptide unit, a hydrophilic aspartic acid-rich peptide unit, and an antibody-binding domain as a functional unit, was constructed. Upon heat induction, ELP-D-C forms micellar nanoparticles displaying antibody-binding domains on their surfaces. The protein nanoparticles were able to incorporate hydrophobic fluorescent compounds and subsequently detect target molecules via antibody binding by the resulting fluorescence intensity, which was proportional to the log of the concentration of the target molecule.

Construction of DNA-NanoLuc luciferase conjugates for DNA aptamer-based sandwich assay using Rep protein.

Objective: We developed a DNA-NanoLuc luciferase (NnaoLuc) conjugates for DNA aptamer-based sandwich assay using the catalytic domain of the replication initiator protein derived from porcine circovirus type 2 (pRep).

Results: For construction of DNA aptamer and NanoLuc conjugate using the catalytic domain of Rep from PCV2. pRep fused to NanoLuc was genetically constructed and expressed in E.

Development of drug-loaded protein nanoparticles displaying enzymatically-conjugated DNA aptamers for cancer cell targeting.

Modification of protein-based drug carriers with tumor-targeting properties is an important area of research in the field of anticancer drug delivery. To this end, we developed nanoparticles comprised of elastin-like polypeptides (ELPs) with fused poly-aspartic acid chains (ELP-D) displaying DNA aptamers. DNA aptamers were enzymatically conjugated to the surface of the nanoparticles via genetic incorporation of Gene A* protein into the sequence of the ELP-D fusion protein.

A DNA-scaffold platform enhances a multi-enzymatic cycling reaction.

Objective: We explored the co-localization of multiple enzymes on a DNA backbone via a DNA-binding protein, Gene-A* (A*-tag) to increase the efficiency of cascade enzymatic reactions.

Results: Firefly luciferase (FLuc) and pyruvate orthophosphate dikinase (PPDK) were genetically fused with A*-tag and modified with single-stranded (ss) DNA via A*-tag. The components were assembled on ssDNA by hybridization, thereby enhancing the efficiency of the cascading bioluminescent reaction producing light emission from pyrophosphate.

Application of elastin-based nanoparticles displaying antibody binding domains for a homogeneous immunoassay.

Nanoparticles are small size-controlled particles from 1 to 100 nm diameters and characterized by their structure, base material and functional units displayed on their surfaces. In this study, protein-based nanoparticles composed of a hydrophobic elastin-like peptide unit, a hydrophilic aspartic acid-rich peptide unit and displaying antibody binding domains on their surfaces, were designed and genetically synthesized. The constituent fusion proteins, termed ELP-D-C, were found to exist in monomeric form (ELP-D-C/monomer) at low temperature.

Design of bFGF-tethered self-assembling extracellular matrix proteins via coiled-coil triple-helix formation.

Self-assembling peptides are attractive materials for tissue engineering applications because of their functionality including high biocompatibility and biodegradability. Modification of self-assembling peptides with functional motifs, such as the cell-adhesive tripeptide sequence RGD leads to functional artificial extracellular matrices (ECMs). In this study, we developed an artificial self-assembling ECM protein tethered with a growth factor via heterotrimer triple-helix (helix A/B/C) formation.

Fluorescent and luminescent fusion proteins for analyses of amyloid beta peptide aggregation.

The amyloid beta (Aβ) peptide is regarded as a causative agent of Alzheimer's disease. In this study, fluorescent and luminescent fusion proteins were constructed to analyze Aβ aggregation. A system was developed to monitor changes in luminescence that provides information about Aβ aggregation.

Design of luciferase-displaying protein nanoparticles for use as highly sensitive immunoassay detection probes.

In this study, we developed a protein nanoparticle-based immunoassay to detect cancer biomarkers using a bioluminescent fusion protein. This method relies on the use of protein nanoparticles comprised of genetically-engineered elastin-like polypeptides (ELPs) fused with poly-aspartic acid tails (ELP-D), previously developed in our lab. The sizes of the self-assembled ELP-D nanoparticles can be regulated at the nanoscale by charged repulsion of the poly-aspartic acid chains.

Development of a Split SNAP-CLIP Double Labeling System for Tracking Proteins Following Dissociation from Protein-Protein Complexes in Living Cells.

The split SNAP-tag protein-fragment complementation assay (PCA) is a useful tool for imaging protein-protein interactions (PPIs) in living cells. In contrast to conventional methods employed for imaging PPIs, the split SNAP-tag PCA enables tracking of proteins following dissociation from protein-protein complexes. A limitation of this system, however, is that it only allows for labeling and tracking of one of the proteins forming the protein-protein complex.

Construction of a Defined Biomimetic Matrix for Long-Term Maintenance of Mouse Induced Pluripotent Stem Cells.

The existing in vitro culture systems often use undefined and animal-derived components for the culture of pluripotent stem cells. Artificial bioengineered peptides have the potential to become alternatives to these components of extracellular matrix (ECM). Integrins and cadherins are two cell adhesion proteins important for stem cell self-renewal, differentiation, and phenotype stability.

Construction of a tissue-specific transcription factor-tethered extracellular matrix protein via coiled-coil helix formation.

Tissue-specific transcription factors are key regulators of cellular differentiation. Previously, we succeeded in introducing basic helix-loop-helix tissue-specific transcription factor proteins into cells to induce cellular differentiation. Based on these results, we decided to focus on the use of tissue-specific transcription factor proteins in the construction of biomaterials.

Delivery of bFGF for Tissue Engineering by Tethering to the ECM.

Delivery of growth factors to target cells is an important subject in tissue engineering. Towards that end, we have developed a growth factor-tethered extracellular matrix (ECM). Here, basic fibroblast growth factor (bFGF) was tethered to extracellular matrix noncovalently.

Growth Factor Tethering to Protein Nanoparticles via Coiled-Coil Formation for Targeted Drug Delivery.

Protein-based nanoparticles are attractive carriers for drug delivery because they are biodegradable and can be genetically designed. Moreover, modification of protein-based nanoparticles with cell-specific ligands allows for active targeting abilities. Previously, we developed protein nanoparticles comprising genetically engineered elastin-like polypeptides (ELPs) with fused polyaspartic acid tails (ELP-D).