Construction of bispecific antibodies by specific pairing between the heavy chain and the light chain using removable SpyCatcher/SnoopCatcher units.

During the production of bispecific antibodies (bsAbs), nonspecific pairing results in low yields of target bsAb molecules, an issue known as the "mispairing problem." Several antibody engineering techniques have been developed to overcome mispairing issues. Here, we introduce "bsAb by external pairing and excision" (BAPE), a novel chain pairing method that induces specific chain pairing by fusing external SpyCatcher/Tag and SnoopCatcher/Tag units.

Domain structure and function of α-1,3-glucanase Agl-EK14 from the gram-negative bacterium Flavobacterium sp. EK-14.

The α-1,3-glucanase Agl-EK14 from Flavobacterium sp. EK-14 comprises a signal peptide (SP), a catalytic domain (CAT), a first immunoglobulin-like domain (Ig1), a second immunoglobulin-like domain (Ig2), a ricin B-like lectin domain (RicinB), and a carboxy-terminal domain (CTD). SP and CTD are predicted to be involved in extracellular secretion, while the roles of Ig1, Ig2, and RicinB are unclear.

Synthesis and evaluation of catecholamine derivatives as amyloid-beta aggregation inhibitors.

Effectively inhibition of amyloid β (Aβ) aggregation is considered an important method for treatment of the Alzheimer's disease. Herein, inspired by the ability of trans-clovamide to effectively inhibit Aβ aggregation, we synthesized a series of structurally related catecholamine derivatives and tested them as Aβ aggregation inhibitors using the Thioflavin T assay. The results show that they demonstrated a higher inhibitory rate against Aβ aggregation.

Redesign of a thioflavin-T-binding protein with a flat β-sheet to evaluate a thioflavin-T-derived photocatalyst with enhanced affinity.

Amyloids, proteinous aggregates with β-sheet-rich fibrils, are involved in several neurodegenerative diseases such as Alzheimer's disease; thus, their detection is critically important. The most common fluorescent dye for amyloid detection is thioflavin-T (ThT), which shows on/off fluorescence upon amyloid binding. We previously reported that an engineered globular protein with a flat β-sheet, peptide self-assembly mimic (PSAM), can be used as an amyloid binding model.

SpyTag Peptide with Alkoxyl Aspartic Acids for pH-Dependent Activation of the SpyCatcher/Tag System.

The SpyCatcher/SpyTag system is a protein pair that forms a covalent isopeptide bond without an additional energy supply. The ability to connect fused proteins makes this system an attractive tool for several protein engineering applications. Conditional activation of the SpyCatcher/SpyTag complex formation further expands the use of this system.

Addition of α-1,3-glucan-binding domains to α-1,3-glucanase Agn1p from Schizosaccharomyces pombe enhances hydrolytic activity of insoluble α-1,3-glucan.

The glycoside hydrolase (GH) 71 α-1,3-glucanase (Agn1p) from Schizosaccharomyces pombe consists of an N-terminal signal sequence and a catalytic domain. Meanwhile, the GH87 α-1,3-glucanase (Agl-KA) from Bacillus circulans KA-304 consists of an N-terminal signal sequence, a first discoidin domain (DS1), a carbohydrate-binding module family 6 (CBM6), a threonine and proline repeat linker (TP), a second discoidin domain (DS2), an uncharacterized domain, and a catalytic domain. DS1, CBM6, and DS2 exhibit α-1,3-glucan binding activity.

β-Strand-mediated Domain-swapping in the Absence of Hydrophobic Core Repacking.

Domain swapping is a process wherein a portion of a protein is exchanged with its counterpart in another copy of the molecule, resulting in the formation of homo-oligomers with concomitant repacking of a hydrophobic core. Here, we report domain swapping triggered upon modifying a β-hairpin sequence within a single-layer β-sheet (SLB) of a model protein, OspA that did not involve the formation of a reorganized hydrophobic core. The replacement of two β-hairpin sequences with a Gly-Gly and shorteing of a β-hairpin resulted in a protein that formed two distinct crystal structures under similar conditions: one was monomeric, similar to the parental molecule, whereas the other was a domain-swapped dimer, mediated by an intermolecular β-sheet in the SLB portion.

α-1,3-Glucanase from the gram-negative bacterium Flavobacterium sp. EK-14 hydrolyzes fungal cell wall α-1,3-glucan.

The glycoside hydrolase (GH) 87 α-1,3-glucanase (Agl-EK14) gene was cloned from the genomic DNA of the gram-negative bacterium Flavobacterium sp. EK14. The gene consisted of 2940 nucleotides and encoded 980 amino acid residues.

Aspergillus oryzae α-l-rhamnosidase: Crystal structure and insight into the substrate specificity.

The subsequent biochemical and structural investigations of the purified recombinant α-l-rhamnosidase from Aspergillus oryzae expressed in Pichia pastoris, designated as rAoRhaA, were performed. The specific activity of the rAoRhaA wild-type was higher toward hesperidin and narirutin, where the l-rhamnose residue was α-1,6-linked to β-d-glucoside, than toward neohesperidin and naringin with an α-1,2-linkage to β-d-glucoside. However, no activity was detected toward quercitrin, myricitrin, and epimedin C.

Construction of IgG-Fab bispecific antibody via intein-mediated protein trans-splicing reaction.

A bispecific antibody (bsAb) is a class of engineered antibody molecules that simultaneously binds to two different antigens by having two kinds of antigen-binding domains. One of the major obstacles for the bsAb production is the incorrect chain-pairing problem, wherein each heavy and light chain should form pairings with the correct counterpart's chains, but the structural similarity of the incorrect partners also forms the incorrect pairings. This study aimed to demonstrate a bsAb construction method using intein-mediated protein trans-splicing to create IgG-Fab-type bsAbs, which is a modified antibody with a structure in which two additional Fabs are linked to the N-terminus of the heavy chain of an IgG molecule.

Incorporation of a repeated polypeptide sequence in therapeutic antibodies as a universal masking procedure: A case study of T cell-engaging bispecific antibodies.

Prodrug design is a promising approach for reducing the off-target effects of therapeutic antibodies, particularly bispecific antibodies (bsAbs) that recruit T cells for activation; this design uses masking sequences that inhibit antibody binding until they reach the tumor microenvironment, where they are removed. In this study, we propose PAS, a polypeptide sequence composed of repeated Pro, Ala, and Ser residues, as a universal masking sequence. PAS has no specificity, but can inhibit antibody binding through steric hindrance caused by its large fluid dynamic radius and disordered structure; additionally, its length can be adjusted.

Heterologous expression of α-1,3-glucanase Agn1p from Schizosaccharomyces pombe, and efficient production of nigero-oligosaccharides by enzymatic hydrolysis from solubilized α-1,3;1,6-glucan.

The glycoside hydrolase family 71 α-1,3-glucanase (Agn1p) of Schizosaccharomyces pombe was expressed in Escherichia coli Rosetta-gami B (DE3). Agn1p (0.5 nmol/mL) hydrolyzed insoluble α-1,3-glucan (1%), and about 3.

Cloning and structural basis of fluorescent protein color variants from identical species of sea anemone, Diadumene lineata.

Diadumene lineata is a colorful sea anemone with orange stripe tissue of the body column and plain tentacles with red lines. We subjected Diadumene lineata to expression cloning and obtained genes encoding orange (OFP: DiLiFP561) and red fluorescent proteins (RFPs: DiLiFP570 and DiLiFP571). These proteins formed obligatory tetramers.

GH-16 Type β-1,3-Glucanase from sp. MK9-1 Enhances Antifungal Activity of GH-19 Type Chitinase, and Its Glucan-binding Domain Binds to Fungal Cell-wall.

The GH-16 type β-1,3-glucanase (BgluC16MK) gene of sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from strain N4-7.

Mutational analysis of the effects of N-glycosylation sites on the activity and thermal stability of rutinosidase from Aspergillus oryzae.

Purified recombinant rutinosidase from Aspergillus oryzae expressed in Pichia pastoris (rAoRutM) exhibits increase in thermal stability after treatment with endo-β-N-acetylglucosaminidase H (endo-H). In this study, the role of N-glycosylation in the activity and thermal stability of rAoRutM was analyzed via site-directed mutagenesis. Based on the crystal structure of AoRutM, five N-glycosylation sites (N32, N128, N176, N288, and N359) were identified in the AoRut protein.

Surface Engineering of Top7 to Facilitate Structure Determination.

Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination.

The total synthesis of berberine and selected analogues, and their evaluation as amyloid beta aggregation inhibitors.

The total synthesis of berberine and selected analogues. And their evaluation as amyloid β (Aβ) aggregation inhibitors is described. The key step in the synthesis, the assembly of the berberine framework, was accomplished using an intermolecular Heck reaction.

Structural analysis of the β-sheet edge of peptide self-assembly using a model protein.

Peptides and proteins self-assemble into β-sheet-rich fibrils, amyloid, which extends its structure by incorporating peptide/protein molecules from solution. At the elongation edge, the peptide/protein molecule binds to the edge of the amyloid β-sheet. Such processes are transient and elusive when observing molecular details by experimental methods.

Cloning, expression, and characterization of a GH 19-type chitinase with antifungal activity from Lysobacter sp. MK9-1.

The chitin-assimilating gram-negative bacterium, Lysobacter sp. MK9-1, was isolated from soil and was the source of a glycoside hydrolase family 19-type chitinase (Chi19MK) gene that is 933-bp long and encodes a 311-residue protein. The deduced amino acid sequence of Chi19MK includes a signal peptide, an uncharacterized sequence, a carbohydrate-binding module family 12-type chitin binding domain, and a catalytic domain.

Aspergillus oryzae Rutinosidase: Biochemical and Structural Investigation.

The rutinosidase (Rut)-encoding gene has been expressed in with its native signal sequence from Biochemical and structural investigation of the purified recombinant mature Rut (Rut), designated rRutM, was performed in this study. A 1.7-Å resolution crystal structure of rRutM was determined, which is an essential step forward in the utilization of Rut as a potential catalyst.

Design, synthesis and evaluation of diaryl γ-dihydropyrone derivatives as cyclocurcumin mimetics and inhibitors of the aggregation of amyloid β.

A structure activity relationship study of cyclocurcumin-derived, diaryl γ-dihydropyrone-based inhibitors of amyloid β aggregation is described. Optimization of the diaryl γ-dihydropyrone framework and two phenolic rings resulted in the identification of diaryl γ-dihydropyrone type cyclocurcumin analogue AY1511, which exhibited potent anti-amyloid β aggregation activity (leading to nanorod-like fragments), sufficient water solubility, and low cytotoxicity.

Residue-Specific Binding Mechanisms of Thioflavin T to a Surface of Flat β-Sheets within a Peptide Self-Assembly Mimic.

Thioflavin T (ThT) is a popular fluorescent dye for detecting amyloid, a protein aggregate with a β-sheet-rich structure that causes many neurodegenerative diseases. Despite the dye's popularity, a detailed understanding of its molecular binding mechanism remains elusive. We previously reported a protein model that can bind ThT on a single-layer β-sheet and revealed that a channel formed by aromatic rings with a confined length enhanced ThT binding.

Cooperative unfolding of a single-layer β-sheet protein, CPAP G-box.

CPAP is a centriolar protein and its C-terminal domain, G-box or TCP, has a very unique structure that comprises a single-layer β-sheet without hydrophobic core packing. Here we characterized its biophysical properties, including its stability against chemical denaturation. Interestingly, upon urea-induced equilibrium unfolding, the CPAP G-box showed cooperative unfolding behavior that is the hallmark of globular proteins.

Molecular basis of flexible peptide recognition by an antibody.

Antibodies can recognize various types of antigens with high specificity and affinity and peptide is one of their major targets. Understanding an antibody's molecular recognition mechanism for peptide is important for developing clones with a higher specificity and affinity. Here, the author reviews recent progresses in flexible peptide recognition by an antibody using several biophysical techniques, including X-ray crystallography, molecular dynamics simulations and calorimetric measurements.