RESUMO
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.
Assuntos
Anticorpos , Engenharia de Proteínas , Aprendizado de MáquinaRESUMO
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.
Assuntos
Biblioteca de Peptídeos , Anticorpos de Domínio Único , Anticorpos , Técnicas de Visualização da Superfície Celular , Regiões Determinantes de Complementaridade , Epitopos , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/químicaRESUMO
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.
Assuntos
Cianobactérias , Peixe-Zebra , Animais , Membrana Celular , Fotossíntese , Bicamadas LipídicasRESUMO
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.
Assuntos
Bacteriófagos , Biblioteca de Peptídeos , Proteínas de Transporte , Teorema de Bayes , Estudos Prospectivos , Bacteriófagos/genética , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
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.
Assuntos
Técnicas Biossensoriais , Glucose Oxidase , Glicemia , Automonitorização da Glicemia , Carbono , Eletrodos , Enzimas Imobilizadas , Flavina-Adenina Dinucleotídeo , Glucose , Glucose 1-Desidrogenase , MucorRESUMO
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.
Assuntos
Saccharomycetales , Técnicas de Inativação de Genes , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Fluxo de TrabalhoRESUMO
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.
Assuntos
Celulases , Proteínas de Escherichia coli , Celulases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucose-6-Fosfato/metabolismo , Periplasma/genéticaRESUMO
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. coli. Nanobody-R9 was synthesized at a maximum binding efficiency of 85.1%, without changing the binding affinity of the nanobody for the antigen. Nanobody-R9 successfully delivered siRNA into the cells, and the cellular influx of siRNA increased with increase in the ratio of Nanobody-R9 to siRNA. We further demonstrated that the Nanobody-R9-siRNA complex, at a 30:1 ratio, induced an approximately 58.6% reduction in the amount of target protein due to RNAi in mRNA compared to lipofectamine.
Assuntos
Sistemas de Liberação de Medicamentos , Peptídeos/química , RNA Interferente Pequeno/administração & dosagem , Anticorpos de Domínio Único/administração & dosagem , Linhagem Celular , Dicroísmo Circular , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Células HEK293 , Humanos , Ligases/metabolismo , Peptídeos/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genéticaRESUMO
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. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.
Assuntos
Conformação Proteica em alfa-Hélice/genética , Engenharia de Proteínas/métodos , Estrutura Secundária de Proteína/genética , Sequência de Aminoácidos/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida/métodos , Desnaturação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína/fisiologia , Proteínas/metabolismoRESUMO
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). We enhanced its cytotoxicity by constructing an Fc fusion protein and rearranging order of the V domain. In this study, we created an additional functional bsAb, by integrating the molecular formats of bsAb and high-affinity mutants previously isolated by phage display in the form of Fv. Introducing the high-affinity mutations into bsDbs successfully increased their affinities and enhanced their cytotoxicity in vitro and in vivo. However, there were some limitations to affinity maturation of bsDb by integrating high-affinity Fv mutants, particularly in Fc-fused bsDb with intrinsic high affinity, because of their bivalency. The tetramers fractionated from the bsDb mutant exhibited the highest in vitro growth inhibition among the small bsAbs and was comparable to the in vivo anti-tumor effects of Fc-fused bsDbs. This molecule shows cost-efficient bacterial production and high therapeutic potential.
Assuntos
Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Complexo CD3/antagonistas & inibidores , Mutação , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologia , Antineoplásicos Imunológicos/química , Complexo CD3/química , Desenho de Fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Ligação Proteica , Engenharia de Proteínas , Proteínas Recombinantes de Fusão , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Fusão Gênica , Neoplasias/tratamento farmacológico , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Linhagem Celular Tumoral , Biologia Computacional , Escherichia coli/genética , Humanos , Modelos Moleculares , Muromonab-CD3/química , Muromonab-CD3/genética , Mutação , Conformação Proteica , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologia , Sítios de Ligação de Anticorpos , Receptores de Antígenos/química , Receptores de Antígenos/imunologia , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Humanos , ProteóliseRESUMO
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 3.7-4.0 eV. In recent years, there have been reports about several ITO-based biosensors. Development of an ITO binding interface for the clustering of sensor proteins without complex bioconjugates is required. OBJECTIVE: In this article, we aimed to identify peptides that bind to indium tin oxide nanoparticles via different binding mechanisms. METHODS: Indium tin oxide nanoparticles binding peptide ware selected using phage display and biopanning against indium tin oxide, under five different buffer conditions and these peptides characterized about binding affinity and specificity. RESULTS: Three types of indium tin oxide nanoparticles-binding peptides were selected from 10 types of peptide candidates identified in phage display and biopanning. These included ITOBP8, which had an acidic isoelectric point, and was identified when a buffer containing guanidine was used, and ITOBP6 and ITOBP7, which contained a His-His-Lys sequence at their N-termini, and were identified when a highly concentrated phosphate elution buffer with a low ionic strength was used. Among these peptides, ITOBP6 exhibited the strongest indium tin oxide nanoparticlesbinding affinity (dissociation constant, 585 nmol/L; amount of protein bound at saturation, 17.5 nmol/m 2 - particles). CONCLUSION: These results indicate that peptides with specific binding properties can be obtained through careful selection of the buffer conditions in which the biopanning procedure is performed.
Assuntos
Oligopeptídeos/análise , Compostos de Estanho/química , Fenômenos Biofísicos , Técnicas Biossensoriais , Soluções Tampão , Nanopartículas , Biblioteca de PeptídeosRESUMO
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 2.73â¯×â¯10-4â¯s-1 (298â¯K) at pH 2.46. The dependence of kdH on the proton concentration (pHâ¯=â¯2.09-3.12) revealed a first-order dependence of kdH on [H+]; kdHâ¯=â¯kd + k1[H+], where kd is the spontaneous dissociation rate and was 5.65â¯×â¯10-5â¯s-1, and k1 is the second-order rate constant and was 5.07â¯×â¯10-2â¯M-1â¯s-1. From the kd value, the half-life of the Try-Apr complex at physiological pH was determined as 3.4â¯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.
Assuntos
Aprotinina/química , Inibidores da Tripsina/química , Tripsina/química , Animais , Ligação Competitiva , Bovinos , Eletroforese Capilar , Meia-Vida , Concentração de Íons de Hidrogênio , Cinética , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
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.
Assuntos
Anticorpos Biespecíficos/química , Desenho de Fármacos , Nanopartículas/química , Nanotecnologia , Catálise , Dimerização , Ouro/química , Fragmentos de Imunoglobulinas/química , Membranas Artificiais , Porosidade , Óxido de Zinco/químicaRESUMO
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.
Assuntos
Ouro , Nanopartículas Metálicas , Coloides , Tamanho da Partícula , ZircônioRESUMO
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. Treatment with 5 nm citrate coated gold nanoparticles recovers the impaired motor stepping by remodeling the A4V SOD1 rather than stabilizing microtubules or protein folding. Instead, gold nanoparticles alter the protein by a mechanism that reforms protein elements of A4V SOD1, in turn fixing the aberrant interaction of kinesin with microtubules. Reinstating kinesin motility holds potential for managing debilitating ALS.
RESUMO
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. In this approach, we conduct two rounds of mutagenesis where an initial library of protein variants is used to train a machine-learning model to guide mutagenesis for the second-round library. This enables us to prepare a small library suited for screening experiments with high enrichment of functional proteins. We demonstrated a proof-of-concept of our approach by altering the reference green fluorescent protein (GFP) so that its fluorescence is changed into yellow. We successfully obtained a number of proteins showing yellow fluorescence, 12 of which had longer wavelengths than the reference yellow fluorescent protein (YFP). These results show the potential of our approach as a powerful method for directed evolution of fluorescent proteins.
Assuntos
Proteínas Luminescentes/genética , Aprendizado de Máquina , Evolução Molecular Direcionada , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , MutagêneseRESUMO
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.
Assuntos
Cinesinas/química , Cinesinas/metabolismo , Microtúbulos/metabolismo , Simulação de Dinâmica Molecular , Rotação , EstereoisomerismoRESUMO
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. However, the affinity was not similar to that of parental 528. For further affinity maturation, we aimed to isolate VL mutants that act in synergy with VH mutants. However, the OS method could not be applied for selecting VL fragments because the preparation of soluble VH fragments was hampered by their instability and insolubility. Therefore, we initially designed a modified OS method based on domain-swapping of VH fragments, from added soluble Fv fragments to phage-displaying VL fragments. Using this novel Fv-added OS selection method, we successfully isolated VL mutants, and one of the Fv comprising VH and VL mutants showed affinity almost equivalent to that of parental 528. This method is applicable for engineering other VL fragments for affinity maturation.