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. We investigated metabolic changes and its mechanisms occurring when specific proteins are localized to the E. coli periplasm. We found that the periplasmic localization of ß-glucosidase promoted the shikimate pathway involved in the synthesis of aromatic chemicals. The periplasmic localization of other proteins with an affinity for glucose-6-phosphate (G6P), such as inactivated mutants of Pgi, Zwf, and PhoA, similarly accelerated the shikimate pathway. Our results indicate that G6P is transported from the cytoplasm to the periplasm by the glucose transporter protein EIICBGlc, and then captured by ß-glucosidase.

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. The circular topology is potentially beneficial for fixing the orientation of two paratopes and protection from exopeptidase digestion. We construct herein a circularly connected bispecific VHH, termed cyclobody, using the split-intein circular ligation of peptides and proteins. The constructed cyclobodies are protected from proteolysis with a retained bispecificity. The anti-EGFR × anti-GFP cyclobody can specifically stain EGFR-positive cells with GFP. The anti-EGFR × anti-CD16 cyclobody shows cytotoxic activity against EGFR-positive cancer cells with comparative activity of a tandem VHH construct. Successful demonstration of a new topology for the bispecific antibody will expand the construction strategy for developing antibody-based drugs and reagents.

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. First, building modules without lysine residues-which can be chemical conjugation sites-were generated by means of genetic mutation. Specific mutated residues in the lysine-free modules were then re-mutated to lysine residues. Chemical conjugation at the recovered lysine sites enabled the construction of homogeneous bivalent and bispecific antibodies from block modules that could not have been so arranged by genetic fusion approaches. Molecular evolution and bioinformatics techniques assisted in finding viable alternatives to the lysine residues that did not deactivate the block modules. Multiple candidates for re-mutation positions offer a wide variety of possible steric arrangements of block modules, and appropriate linkages between block modules can generate highly bioactive bispecific antibodies. Here, we propose the effectiveness of the lysine-free block module design for site-specific chemical conjugation to form a variety of types of homogeneous chimeric protein-format molecule with a finely tuned structure and function.

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. By analyzing the dependence of peak height of Try-Apr on separation time, the dissociation rate kdH was obtained as 2.73 × 10-4 s-1 (298 K) at pH 2.46. The dependence of kdH on the proton concentration (pH = 2.09-3.12) revealed a first-order dependence of kdH on [H+]; kdH = kd + k1[H+], where kd is the spontaneous dissociation rate and was 5.65 × 10-5 s-1, and k1 is the second-order rate constant and was 5.07 × 10-2 M-1 s-1. From the kd value, the half-life of the Try-Apr complex at physiological pH was determined as 3.4 h. The presence of the proton-assisted dissociation can be explained by the protonation of -COO- of the Asp residue in Try, which breaks the salt bridge with the -NH3+ group of Lys in Apr.

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. The relatively low melting point of the gold nanoparticles and the solubility of ZnO in dilute acidic solution then allowed for the bottom-up synthesis of a nanoporous gold membrane by means of a low-energy, low-environmental-load protocol. The nanoporous gold membrane showed high catalytic activity for the reduction of p-nitrophenol to p-aminophenol by sodium borohydride. Here, we show the potential utility of nanoparticle pairing mediated by bispecific antibodies for the bottom-up construction of nanostructured materials from multiple nanomaterials.

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, Equisetum arvense 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. ζ-potential measurement indirectly determined nanoparticles stability in liquid medium. Ultraviolet-Visible Spectroscopy characterized basic absorbance maxima for both Au and the bi-phasic Au/ZrO2 system. Finally, total metal concentration was determined using Inductively Coupled Plasma Mass Spectrometry. ζ-potential measurements proved satisfactory stability of both Au (-13.4 to -17 mV) and Au/ZrO2 nanoparticles (-14.1 to -17.5 mV) over the experimental period. Scanning Transmission Electron Microscopy with Selected Area Diffraction analysis confirmed nanoparticles crystalline nature, and we determined 24 nm and 40 nm core nanogold diameters in Au and Au/ZrO2 nanoparticle colloids. Dynamic light scattering analysis confirmed the dichotomy between particle sizes in liquid medium in the hundreds of nanometers measured, and long-term measurements confirmed reasonable colloid stability-a paramount parameter for potential nanoparticles applications; especially in heterogeneous catalysis.

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. However, to date there lacks convincing evidence of a hierarchical connection of the molecular nature of microtubules to large-scale chirality, particularly at the length scale of an entire cell. Here we assemble an in vitro active layer, consisting of microtubules and kinesin motor proteins, on a glass surface. Upon inclusion of methyl cellulose, the layered system exhibits a long-range active nematic phase, characterized by the global alignment of gliding MTs. This nematic order spans over the entire system size in the millimeter range and, remarkably, allows hidden collective chirality to emerge as counterclockwise global rotation of the active MT layer. The analysis based on our theoretical model suggests that the emerging global nematic order results from the local alignment of MTs, stabilized by methyl cellulose. It also suggests that the global rotation arises from the MTs' intrinsic curvature, leading to preferential handedness. Given its flexibility, this layered in vitro cytoskeletal system enables the study of membranous protein behavior responsible for important cellular developmental processes.

Salt-Switchable Artificial Cellulase Regulated by a DNA Aptamer.

A novel artificial cellulase was developed by conjugating a DNA aptamer to an endoglucanase catalytic domain, thereby substituting the natural carbohydrate-binding module. Circular dichroism spectroscopy and adsorption isotherm showed the binding motif of cellulose-binding DNA aptamer (CelApt) was G-quadruplex and stem-loop structures stabilized in the presence of salts, and CelApt binding preferred the amorphous region of the solid cellulose. By introducing the revealed salt-switchable cellulose-binding nature of CelApt into a catalytic domain of a cellulase, we created CelApt-catalytic domain conjugate possessing both controllable adsorption on the solid substrates and equal enzymatic activity to the wild-type cellulase. Thus potential use of a responsive DNA aptamer for biocatalysis at a solid surface was demonstrated.

Microtubule guiding in a multi-walled carbon nanotube circuit.

In nanotechnological devices, mass transport can be initiated by pressure driven flow, diffusion or by employing molecular motors. As the scale decreases, molecular motors can be helpful as they are not limited by increased viscous resistance. Moreover, molecular motors can move against diffusion gradients and are naturally fitted for nanoscale transportation. Among motor proteins, kinesin has particular potential for lab-on-a-chip applications. It can be used for sorting, concentrating or as a mechanical sensor. When bound to a surface, kinesin motors propel microtubules in random directions, depending on their landing orientation. In order to circumvent this complication, the microtubule motion should be confined or guided. To this end, dielectrophoretically aligned multi-walled-carbon nanotubes (MWCNT) can be employed as nanotracks. In order to control more precisely the spatial repartition of the MWCNTs, a screening method has been implemented and tested. Polygonal patterns have been fabricated with the aim of studying the guiding and the microtubule displacement between MWCNT segments. Microtubules are observed to transfer between MWCNT segments, a prerequisite for the guiding of microtubules in MWCNT circuit-based biodevices. The effect of the MWCNT organization (crenellated or hexagonal) on the MT travel distance has been investigated as well.

Molecular motor-powered shuttles along multi-walled carbon nanotube tracks.

As a complementary tool to nanofluidics, biomolecular-based transport is envisioned for nanotechnological devices. We report a new method for guiding microtubule shuttles on multi-walled carbon nanotube tracks, aligned by dielectrophoresis on a functionalized surface. In the absence of electric field and in fluid flow, alignment is maintained. The directed translocation of kinesin propelled microtubules has been investigated using fluorescence microscopy. To our knowledge, this is the first demonstration of microtubules gliding along carbon nanotubes.

Microtubule shuttles on kinesin-coated glass micro-wire tracks.

Gliding of microtubule filaments on surfaces coated with the motor protein kinesin has potential applications for nano-scale devices. The ability to guide the gliding direction in three dimensions allows the fabrication of tracks of arbitrary geometry in space. Here, we achieve this by using kinesin-coated glass wires of micrometer diameter range. Unlike previous methods in which the guiding tracks are fixed on flat two-dimensional surfaces, the flexibility of glass wires in shape and size facilitates building in-vitro devices that have deformable tracks.

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. In our approach, multiple prediction models are trained by subsampling the training set and the multi-objective optimization problem, where one objective is the average activity and the other is the standard deviation, is solved. The Pareto front represents a list of sequences with the whole spectrum of activity and stability. Using this method, we designed VHH (Variable domain of Heavy chain of Heavy chain) antibodies based on the dataset obtained from deep mutational screening. To solve multi-objective optimization, we employed our sequence design software MOQA that uses quantum annealing. By applying several selection criteria to 19,778 designed sequences, five sequences were selected for wet-lab validation. One sequence, 16 mutations away from the closest training sequence, was successfully expressed and found to possess desired binding specificity. Our whole spectrum approach provides a balanced way of dealing with the prediction uncertainty, and can possibly be applied to extensive search of functional sequences.

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. The clustering analysis of the deep sequencing data from appropriate steps revealed no distinct sequence patterns, but a Bayesian machine learning model trained with the selected deep sequencing data supplied nine clusters with distinct sequence patterns. Phage libraries were designed on the basis of the sequence patterns identified, and four improved variants with target-specific affinity (EC50 = 80-277 nM) were identified by biopanning. The selection and use of deep sequencing data without undesirable bias enabled us to extract the information on prospective variants. In summary, the use of appropriate deep sequencing data and machine learning with the sequence data has the possibility of finding sequence space where functional variants are enriched.

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. First, membrane dynamics of a lipid bilayer for the cell membrane is described. Since the cell membrane separates the inside of the cell from the outside, self-organizing systems that form spatial patterns on membranes often depend on non-linear dynamics. Second, various data banks based on recent genomics analysis supply the data including vast functional proteins from many organisms and their variable species. Since the proteins existing in nature are only a very small part of the space represented by amino acid sequence, success of mutagenesis-based molecular evolution approach crucially depends on preparing a library with high enrichment of functional proteins. Third, photosynthetic organisms depend on ambient light, the regular and irregular changes of which have a significant impact on photosynthetic processes. The light-driven process proceeds through many redox couples in the cyanobacteria constituting chain of redox reactions. The fourth topic focuses on a vertebrate model, the zebrafish, which can help to understand, predict and control the chaos of complex biological systems. In particular, during early developmental stages, developmental differentiation occurs dynamically from a fertilized egg to divided and mature cells. These exciting fields of complexity, chaos, and non-linear science have experienced impressive growth in recent decades. Finally, future directions for non-linear biosystems are presented.

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. The binding-site sequence in a native protein was grafted into a complementarity-determining region (CDR) in the nanobody, and a nonrelated CDR loop (in the grafted nanobody) was randomized to create a phage display library. In this construction of nanobodies by integrating graft and evolution technology (CAnIGET method), suitable grafting of the functional sequence added functionality to the nanobody, and the molecular-evolution approach enhanced the binding function to inhibit the native protein-protein interactions. To apply for biological tool with growth screening, model nanobodies with an affinity for filamenting temperature-sensitive mutant Z (FtsZ) from Staphylococcus aureus were constructed and completely inhibited the polymerization of FtsZ as a function. Consequently, the expression of these nanobodies drastically decreased the cell division rate. We demonstrate the potential of the CAnIGET method with the use of native protein-protein interactions for steady epitope-specific evolutionary engineering.

Cytotoxic enhancement of a bispecific diabody by format conversion to tandem single-chain variable fragment (taFv): the case of the hEx3 diabody.

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.

A high-affinity gold-binding camel antibody: antibody engineering for one-pot functionalization of gold nanoparticles as biointerface molecules.

Antibodies, with their high affinity and specificity, are widely utilized in the field of protein engineering, medicinal chemistry, and nanotechnology applications, and our recent studies have demonstrated the recognition and binding of antibody for the surface on inorganic material. In this study, we generated a high-affinity gold-binding antibody fragment by a combination of peptide-grafting and phage-display techniques and showed the availability of the material-binding fragment for one-pot functionalization of nanoparticles as interface molecules. After a gold-binding peptide sequence was grafted into one of the complementarity determining regions of a single variable domain of a heavy-chain camel antibody, a combinatorial library approach raised by 20 times the affinity of the peptide-grafted fragment. The high-affinity gold-binding fragment (E32) spontaneously adsorbed on gold nanoparticles, and consequently the nanoparticles formed a stable dispersion in a high-ionic-strength solution. Multivalent and bispecific antibodies constructed on the E32 platform by means of fusion technology functionalized gold nanoparticles in one pot, and these functionalized nanoparticles could be used to obtain surface plasmon resonance scattering images of cancer cells and to spontaneously link two different nanomaterials. Here, we propose the bispecific antibodies as convenient interface molecules in the nanosized world.

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 (Tm) values and by short heat treatment but not at around 37 °C. Glucose oxidase (GOD) is used in current CGM sensors. In this study, we evaluated the stability of modified Mucor-derived flavin adenine dinucleotide-dependent glucose dehydrogenase (designated Mr144-297) with improved thermal stability at medium to high temperatures and compared it with that of GOD. The Tm value of Mr144-297 was 61.6 ± 0.3 °C and was similar to that of GOD (61.4 ± 1.2 °C). However, Mr144-297 was clearly more stable than GOD at 40 °C and 55 °C. At 37 °C, the stability of a carbon electrode with immobilized Mr144-297 was higher than that of an electrode with GOD. Our data indicate that Mr144-297 is a more suitable enzyme for CGM sensors than is GOD.

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. Using the consolidated screening system, we evaluated >19,000 mutant strains from a mutant library prepared by a modified random gene-disruption method, and identified six factors for which disruption led to increased antibody production. We then combined the disruptions, up to quadruple gene knockouts, which appeared to contribute independently, in a single strain and observed an additive effect. Target protein and promoter were basically interchangeable for the effects of knockout genes screened. We finally used adaptive evolution to recover reduced cell growth by multiple gene knockouts and examine the possibility for further enhancing protein secretion. Our successful, three-part approach holds promise as a method for improving protein production by non-conventional microorganisms.

High affinity anti-inorganic material antibody generation by integrating graft and evolution technologies: potential of antibodies as biointerface molecules.

Recent advances in molecular evolution technology enabled us to identify peptides and antibodies with affinity for inorganic materials. In the field of nanotechnology, the use of the functional peptides and antibodies should aid the construction of interface molecules designed to spontaneously link different nanomaterials; however, few material-binding antibodies, which have much higher affinity than short peptides, have been identified. Here, we generated high affinity antibodies from material-binding peptides by integrating peptide-grafting and phage-display techniques. A material-binding peptide sequence was first grafted into an appropriate loop of the complementarity determining region (CDR) of a camel-type single variable antibody fragment to create a low affinity material-binding antibody. Application of a combinatorial library approach to another CDR loop in the low affinity antibody then clearly and steadily promoted affinity for a specific material surface. Thermodynamic analysis demonstrated that the enthalpy synergistic effect from grafted and selected CDR loops drastically increased the affinity for material surface, indicating the potential of antibody scaffold for creating high affinity small interface units. We show the availability of the construction of antibodies by integrating graft and evolution technology for various inorganic materials and the potential of high affinity material-binding antibodies in biointerface applications.