Abstract Nanobodies (Nbs) hold great potential to replace conventional antibodies in various biomedical applications. However, methods for their discovery can be time-consuming and expensive. We have developed a reliable protein selection strategy that combines magnetic activated cell sorting (MACS)-based screening of yeast surface display (YSD) libraries functional ligand-binding identification by Tat-based recognition associating proteins (FLI-TRAP) isolate antigen-specific Nbs from...

Improving the affinity of protein-protein interactions is a challenging problem that particularly important in development antibodies for diagnostic and clinical use. Here, we used structure-based computational methods to optimize binding VHNAC1, single-domain intracellular antibody (intrabody) from camelid family was selected its specific nonamyloid component (NAC) human α-synuclein (α-syn), natively disordered protein, implicated pathogenesis Parkinson's disease (PD) related neurological...

We have developed a reliable genetic selection strategy for isolating interacting proteins based on the “hitchhiker” mechanism of Escherichia coli twin-arginine translocation (Tat) pathway. This method, designated FLI-TRAP (functional ligand-binding identification by Tat-based recognition associating proteins), is unique ability Tat system to efficiently cotranslocate noncovalent complexes 2 folded polypeptides. In assay, protein be screened interactions engineered with an N-terminal signal...

The bacterial twin-arginine translocation (Tat) pathway facilitates the transport of correctly folded proteins across tightly sealed cytoplasmic membrane. Here, we report isolation and characterization suppressor mutations in Tat translocase that allow export misfolded proteins, which form structures are not normally tolerated by wild-type translocase. Selection suppressors was enabled a genetic assay effectively linked vivo folding stability test protein with efficiency selectable marker...

The 'hitchhiker' mechanism of the bacterial twin-arginine translocation pathway has previously been adapted as a genetic selection for detecting pairwise protein interactions in cytoplasm living Escherichia coli cells. Here, we extended this method, called FLI-TRAP, rapid isolation intracellular antibodies (intrabodies) single-chain Fv format that possess superior traits simply by demanding growth on high concentrations antibiotic. Following just single round survival-based enrichment using...

Ascorbic acid has many benefits to the skin. Numerous attempts promote its topical delivery show great challenges since chemical instability and poor skin impermeability. Microneedle is a simple, safe, painless effective means deliver therapeutic or nourishing molecules into The purpose of this study was twofold: (a) develop new formulation ascorbic acid-loaded microneedles enhance stability by investigating an optimal amount polyethyleneimine as additive dextran-based microneedle (b) assess...

The ongoing COVID-19 pandemic has resulted from widespread infection by the SARS-CoV-2 virus. As new variants of concern continue to emerge, understanding correlation between level neutralizing antibodies (NAb) and clinical protection SAR-CoV-2 could be critical in planning next steps vaccine programs. This study explored potential usefulness E. coli as an alternative expression system that can used produce a receptor-binding domain (RBD) for development affordable flexible NAb detection...

Abstract There is an urgent need for affinity reagents that target phospho-modified sites on individual proteins; however, generating such remains a significant challenge. Here, we describe genetic selection strategy routine laboratory isolation of phospho-specific designed ankyrin repeat proteins (DARPins) by linking in vivo capture phosphorylated protein with antibiotic resistance Escherichia coli cells. The assay validated using existing panel DARPins selectively bind the...

Escherichia coli remains one of the preferred hosts for biotechnological protein production due to its robust growth in culture and ease genetic manipulation. It is often desirable export recombinant proteins into periplasmic space reasons related proper disulfide bond formation, prevention aggregation proteolytic degradation, purification. One such system expressing heterologous secreted twin-arginine translocation (Tat) pathway, which has unique advantage delivering correctly folded...

This study reports the development of an amperometric biosensor for quantification mevalonic acid (MA), which is first intermediate HMG-CoA reductase in isoprenoids biosynthesis pathway and therefore a useful indicator HMGR activity. method offers important advantages over previous because no radiolabeled substrates or expensive techniques are required, time analysis relatively short. Self-assembled NAD+ onto multiwall-carbon nanotubes (MWCNTs) was synthesized biosensing system. Adsorption...

Natural rubber (NR) composite foam was developed through the Dunlop process with better volatile solvent absorption and antibacterial activity.The unmodified modified titanium dioxide (TiO 2 ), namely TiO -zinc oxide -ZnO) -silver doped zirconium -Ag-Zr), were incorporated into foams.The photocatalytic origination under UV visible light leads -ZnO to exhibit effective suitable benzene absorbing removing abilities.Thus, specific sizes provided gaseous of 14.0 ppm within 3 h elimination 7.6 14...

Monoclonal antibodies (mAbs) are one of the fastest-growing areas biopharmaceutical industry and have been widely used for a broad spectrum diseases. Meanwhile, immunogenicity non-human derived can generate side effects by inducing human immune response to produce anti-mouse-immunoglobulin antibody (HAMA). In this work, we aim reduce muMAb A.4.6.1 substitute sequences murine sequences. Humanized constructed grafting, specificity determining residues (SDR), complementary regions (CDR),...

Abstract Floral scents are used in many industries and demand for them increases every year. However, the shortage of extracted fragrance supplies from plants leads to high market costs floral scents. Production these fragrances microorganisms is an alternative. In this work we applied synthetic biology approach construct a Escherichia coli system produce linalool, which main component The E. was synthesized laboratory by constructing 2 new BioBrick parts, GPPS LIS Bricks. They were employed...

Interacting RNA-based devices have increasingly gained interest among synthetic biologists due to their potential applications in biotechnology and medicine. However, the effort create novel interacting has been hampered by difficulty designing efficient RNA molecules with desired structures functions. In this work, we propose a new computational platform that efficiently designs RNAs can fold into specific shapes. This combines domain-based decomposition (iDoDe) Domain-based Design (DD)...

A key goal of synthetic biology is to use biological molecules create novel systems. Due their role as transmitters in such systems, RNA have gained much attention from biologists design and construct with desirable functions properties. In recent decades, the RNAs, however, has been limited architecture primitive functions: aptamer catalysis. To expand paradigm RNA-based design, we herein propose a conceptual RNA-RNA interaction based considering domain-based structures well thermodynamic...

The urgent need for an effective COVID-19 therapy has propelled the exploration of innovative strategies to combat fast-mutating SARS-CoV-2 virus. This study attempted develop nanobodies (Nbs) against Omicron variants by redirecting 1.29 neutralizing Nb, a receptor-binding domain (RBD)-specific Nb that can protect various other than Omicron, target subvariant BA.5, variant used development bivalent vaccine. Error-prone libraries were constructed. Following two rounds selection using...

Linalool is a major volatile compound used frequently as an ingredient of perfumes and fragrances. Typically, it produced by chemical routes that are not eco-friendly. We have previously succeeded to construct synthetic Escherchia coli producing linalool. However, the linalool productivity this strain economically feasible. In work, metabolic network E. was analyzed using constraint-based modeling through flux balance analysis (FBA) single gene deletion enhance production. The results...

Protease inhibitors (PIs) have been used to treat various types of symptoms or diseases. However, current PIs block the protease activity by targeting active site which has shown be sensitive off-target effect due crossreactivity with homologues. An alternative approach inhibiting is target substrate, specifically blocking substrate cleavage site. We propose employ synthetic biology create a E. coli as inhibitor detecting biomachine that can effectively isolate intrabodies, new generation...

Monoclonal antibody (mAb) is a very useful tool in not only medical field but also research field. However, the current cost of its production high as most mAbs are produced mammalian cell system. Escherichia coli good alternative has many bottlenecks. Recently, it was demonstrated that biological active full-length Immunoglobulin G (IgG) could be reductive cytoplasm an engineered E. strain called SHuffle. In this study, we investigated effect temperature and induction level on humanized...

Abstract The twin-arginine translocation (Tat) pathway involves an inbuilt quality control (QC) system that synchronizes proofreading of substrate protein folding with lipid bilayer transport. However, the molecular details this QC mechanism remain poorly understood. Here, we hypothesized conformational state Tat substrates is directly sensed by TatB component bacterial translocase. In support hypothesis, several variants in which cytoplasmic membrane-extrinsic domain was either truncated or...

Abstract Monoclonal antibodies (mAbs) have been used extensively both for treatment and diagnostics. Phage display has successfully isolation of many mAbs currently sold in the market. However, main drawback is that it could result a large number false positives. In this study, we explored feasibility combination two powerful antibody techniques, phage Functional L igand-binding Identification by T at-based R ecognition A ssociating P roteins (FLI-TRAP), to identify nanobodies (Nbs) are...