Combinatorial optimization of the hybrid cellulase complex structure designed from modular libraries.

Cellulase selectively recognizes cellulose surfaces and cleaves their β-1,4-glycosidic bonds. Combining hydrolysis using cellulase and fermentation can produce alternative fuels and chemical products. However, anaerobic bacteria produce only low levels of highly active cellulase complexes so-called cellulosomes.

Extensive antibody search with whole spectrum black-box optimization.

In designing functional biological sequences with machine learning, the activity predictor tends to be inaccurate due to shortage of data. Top ranked sequences are thus unlikely to contain effective ones. This paper proposes to take prediction stability into account to provide domain experts with a reasonable list of sequences to choose from.

Synthesis of epitope-targeting nanobody based on native protein-protein interactions for FtsZ filamentation suppressor.

Phage display and biopanning are powerful tools for generating binding molecules for a specific target. However, the selection process based only on binding affinity provides no assurance for the antibody's affinity to the target epitope. In this study, we propose a molecular-evolution approach guided by native protein-protein interactions to generate epitope-targeting antibodies.

Recent research advances on non-linear phenomena in various biosystems.

All biological phenomena can be classified as open, dissipative and non-linear. Moreover, the most typical phenomena are associated with non-linearity, dissipation and openness in biological systems. In this review article, four research topics on non-linear biosystems are described to show the examples from various biological systems.

Selection of target-binding proteins from the information of weakly enriched phage display libraries by deep sequencing and machine learning.

Despite the advances in surface-display systems for directed evolution, variants with high affinity are not always enriched due to undesirable biases that increase target-unrelated variants during biopanning. Here, our goal was to design a library containing improved variants from the information of the "weakly enriched" library where functional variants were weakly enriched. Deep sequencing for the previous biopanning result, where no functional antibody mimetics were experimentally identified, revealed that weak enrichment was partly due to undesirable biases during phage infection and amplification steps.

Comparison of the stability of Mucor-derived flavin adenine dinucleotide-dependent glucose dehydrogenase and glucose oxidase.

Long-term stability at near-body temperature is important for continuous glucose monitoring (CGM) sensors. However, the stability of enzymes used in CGM sensors has often been evaluated by measuring their melting temperature (T) values and by short heat treatment but not at around 37 °C. Glucose oxidase (GOD) is used in current CGM sensors.

A streamlined strain engineering workflow with genome-wide screening detects enhanced protein secretion in Komagataella phaffii.

Expression of secreted recombinant proteins burdens the protein secretion machinery, limiting production. Here, we describe an approach to improving protein production by the non-conventional yeast Komagataella phaffii comprised of genome-wide screening for effective gene disruptions, combining them in a single strain, and recovering growth reduction by adaptive evolution. For the screen, we designed a multiwell-formatted, streamlined workflow to high-throughput assay of secretion of a single-chain small antibody, which is cumbersome to detect but serves as a good model of proteins that are difficult to secrete.

G6P-capturing molecules in the periplasm of Escherichia coli accelerate the shikimate pathway.

Escherichia coli, the most studied prokaryote, is an excellent host for producing valuable chemicals from renewable resources as it is easy to manipulate genetically. Since the periplasmic environment can be easily controlled externally, elucidating how the localization of specific proteins or small molecules in the periplasm affects metabolism may lead to bioproduction development using E. coli.

Enzymatic ligation of an antibody and arginine 9 peptide for efficient and cell-specific siRNA delivery.

A fusion protein comprising an antibody and a cationic peptide, such as arginine-9 (R9), is a candidate molecule for efficient and cell-specific delivery of siRNA into cells in order to reduce the side effects of nucleic acid drugs. However, their expression in bacterial hosts, required for their development, often fails, impeding research progress. In this study, we separately prepared anti-EGFR nanobodies with the K-tag sequence MRHKGS at the C-terminus and R9 with the Q-tag sequence LLQG at the N-terminus, and enzymatically ligated them in vitro by microbial transglutaminase to generate Nanobody-R9, which is not expressed as a fused protein in E.

Rational thermostabilisation of four-helix bundle dimeric de novo proteins.

The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations.

Build-up functionalization of anti-EGFR × anti-CD3 bispecific diabodies by integrating high-affinity mutants and functional molecular formats.

Designing non-natural antibody formats is a practical method for developing highly functional next-generation antibody drugs, particularly for improving the therapeutic efficacy of cancer treatments. One approach is constructing bispecific antibodies (bsAbs). We previously reported a functional humanized bispecific diabody (bsDb) that targeted epidermal growth factor receptor and CD3 (hEx3-Db).

Chemically Crosslinked Bispecific Antibodies for Cancer Therapy: Breaking from the Structural Restrictions of the Genetic Fusion Approach.

Antibodies are composed of structurally and functionally independent domains that can be used as building blocks to construct different types of chimeric protein-format molecules. However, the generally used genetic fusion and chemical approaches restrict the types of structures that can be formed and do not give an ideal degree of homogeneity. In this study, we combined mutation techniques with chemical conjugation to construct a variety of homogeneous bivalent and bispecific antibodies.

Construction of a circularly connected VHH bispecific antibody (cyclobody) for the desirable positioning of antigen-binding sites.

A bispecific antibody (bsAb) is an emerging class of next-generation biological therapeutics. BsAbs are engineered antibodies possessing dual antigen-binding paratopes in one molecule. The circular backbone topology has never been demonstrated, although an enormous number of bispecific constructs have been proposed.

Identification of Indium Tin Oxide Nanoparticle-Binding Peptides via Phage Display and Biopanning Under Various Buffer Conditions.

Background: By recent advances in phage-display approaches, many oligopeptides exhibiting binding affinities for metal oxides have been identified. Indium tin oxide is one of the most widely used conductive oxides, because it has a large band gap of 3.7-4.

Nanoparticle Assisted Remodeling of Proteotoxic SOD1 Mutants Alters the Biointerface of the Functional Interaction of Microtubules and Kinesin Motors.

Transport deficits with motor neuron degeneration have been implicated in amyotrophic lateral sclerosis (ALS). We report a biomimetic system composed of microtubules/kinesin motor that mimics the dysregulated motor dynamics of ALS. Pathogenic ALS mutants A4V SOD1 completely shut off motility.

Capillary electrophoretic reactor for estimation of spontaneous dissociation rate of Trypsin-Aprotinin complex.

A capillary electrophoretic reactor was used to analyze the dissociation kinetics of an enzyme-inhibitor complex in a homogeneous solution without immobilization. The complex consisting of trypsin (Try) and aprotinin (Apr) was used as the model. Capillary electrophoresis provided a reaction field for Try-Apr complex to dissociate through the steady removal of free Try and Apr from the Try-Apr zone.

Complementary Design for Pairing between Two Types of Nanoparticles Mediated by a Bispecific Antibody: Bottom-Up Formation of Porous Materials from Nanoparticles.

Recent advances in biotechnology have enabled the generation of antibodies with high affinity for the surfaces of specific inorganic materials. Herein, we report the synthesis of functional materials from multiple nanomaterials by using a small bispecific antibody recombinantly constructed from gold-binding and ZnO-binding antibody fragments. The bispecific antibody-mediated spontaneous linkage of gold and ZnO nanoparticles forms a binary gold-ZnO nanoparticle composite membrane.

Phytosynthesis of Au and Au/ZrO₂ bi-Phasic System Nanoparticles with Evaluation of Their Colloidal Stability.

Due to its easy availability, preparation, handling and non-toxic nature, horsetail extract was chosen as a reducing, stabilizing and functionalizing agent for Au and bi-phasic Au/ZrO₂ nanoparticle phytosynthesis-inorganic nanoparticle synthesis mediated by plant extract. We studied Au and bi-phasic Au/ZrO₂ nanoparticles in colloids by various physical-chemical and analytical methods over 5 weeks. Dynamic Light Scattering and Scanning Transmission Electron Microscopy compared core and hydrodynamic diameters of nanoparticles.

Machine-Learning-Guided Mutagenesis for Directed Evolution of Fluorescent Proteins.

Molecular evolution based on mutagenesis is widely used in protein engineering. However, optimal proteins are often difficult to obtain due to a large sequence space. Here, we propose a novel approach that combines molecular evolution with machine learning.

Large-scale chirality in an active layer of microtubules and kinesin motor proteins.

During the early developmental process of organisms, the formation of left-right laterality requires a subtle mechanism, as it is associated with other principal body axes. Any inherent chiral feature in an egg cell can in principal trigger this spontaneous breaking of chiral symmetry. Individual microtubules, major cytoskeletal filaments, are known as chiral objects.

Affinity maturation of humanized anti-epidermal growth factor receptor antibody using a modified phage-based open sandwich selection method.

Affinity maturation is one of the cardinal strategies for improving antibody function using in vitro evolutionary methods; one such well-established method is phage display. To minimise gene deletion, we previously developed an open sandwich (OS) method wherein selection was performed using only phage-displaying VH fragments after mixing with soluble VL fragments. The decrease in anti-EGFR antibody 528 affinity through humanization was successfully recovered by selecting VH mutants using this OS method.

Structural considerations for functional anti-EGFR × anti-CD3 bispecific diabodies in light of domain order and binding affinity.

We previously reported a functional humanized bispecific diabody (bsDb) that targeted EGFR and CD3 (hEx3-Db) and enhancement of its cytotoxicity by rearranging the domain order in the V domain. Here, we further dissected the effect of domain order in bsDbs on their cross-linking ability and binding kinetics to elucidate general rules regarding the design of functional bsDbs. Using Ex3-Db as a model system, we first classified the four possible domain orders as anti-parallel (where both chimeric single-chain components are variable heavy domain (VH)-variable light domain (VL) or VL-VH order) and parallel types (both chimeric single-chain components are mixed with VH-VL and VL-VH order).

High-throughput cytotoxicity and antigen-binding assay for screening small bispecific antibodies without purification.

The cytotoxicity of T cell-recruiting antibodies with their potential to damage late-stage tumor masses is critically dependent on their structural and functional properties. Recently, we reported a semi-high-throughput process for screening highly cytotoxic small bispecific antibodies (i.e.

Impact in stability during sequential CDR grafting to construct camelid VHH antibodies against zinc oxide and gold.

Biomolecules which recognize inorganic materials and metal surfaces gain much attention for creating new type of nanomaterials and sensors. 4F2, a camelid VHH antibody, recognizes ZnO surface and has been applied for sensor applications. 4F2 was constructed sequential complementarity determining region (CDR) replacement on the parental VHH antibody, termed the Construction of Antibody by Integrating Grafting and Evolution Technology; CAnIGET procedure.

Use of a Phage-Display Method to Identify Peptides that Bind to a Tin Oxide Nanosheets.

Background: Nanosheets of SnO2 which an n-type semiconductor with a rutile-type crystalline structure are predominantly used as gas sensors. SnO2 nanosheets have a tetragonal crystal structure where growth along the c-axis is suppressed to form a sheet. The major exposed facets of SnO2 nanosheets have {110}, {101} and {211} crystal planes along the a-axis, with the reduced {110} surface having a particularly high surface energy.