Engineering Stem Cell Fate Controlling Biomaterials to Develop Muscle Connective Tissue Layered Myofibers.

Skeletal muscle connective tissue (MCT) surrounds myofiber bundles to provide structural support, produce force transduction from tendons, and regulate satellite cell differentiation during muscle regeneration. Engineered muscle tissue composed of myofibers layered within MCT has not yet been developed. Herein, a bioengineering strategy to create MCT-layered myofibers through the development of stem cell fate-controlling biomaterials that achieve both myogenesis and fibroblast differentiation in a locally controlled manner at the single construct is introduced. The reciprocal role of transforming growth factor-beta 1 (TGF-ß1) and its inhibitor as well as 3D matrix stiffness to achieve co-differentiation of MCT fibroblasts and myofibers from a human-induced pluripotent stem cell (hiPSC)-derived paraxial mesoderm is studied. To avoid myogenic inhibition, TGF-ß1 is conjugated on the gelatin-based hydrogel to control the fibroblasts' populations locally; the TGF-ß1 degrades after 2 weeks, resulting in increased MCT-specific extracellular matrix (ECM) production. The locations of myofibers and fibroblasts are precisely controlled by using photolithography and co-axial wet spinning techniques, which results in the formation of MCT-layered functional myofibers in 3D constructs. This advanced engineering strategy is envisioned as a possible method for obtaining biomimetic human muscle grafts for various biomedical applications.

Direct Recovery of the Rare Earth Elements Using a Silk Displaying a Metal-Recognizing Peptide.

Rare earth elements (RE) are indispensable metallic resources in the production of advanced materials; hence, a cost- and energy-effective recovery process is required to meet the rapidly increasing RE demand. Here, we propose an artificial RE recovery approach that uses a functional silk displaying a RE-recognizing peptide. Using the piggyBac system, we constructed a transgenic silkworm in which one or two copies of the gene coding for the RE-recognizing peptide (Lamp1) was fused with that of the fibroin L (FibL) protein. The purified FibL-Lamp1 fusion protein from the transgenic silkworm was able to recognize dysprosium (Dy3+), a RE, under physiological conditions. This method can also be used with silk from which sericin has been removed. Furthermore, the Dy-recovery ability of this silk was significantly improved by crushing the silk. Our simple approach is expected to facilitate the direct recovery of RE from an actual mixed solution of metal ions, such as seawater and industrial wastewater, under mild conditions without additional energy input.

Site-Specific Chemical Conjugation of Antibodies by Using Affinity Peptide for the Development of Therapeutic Antibody Format.

Artificially modified IgG molecules are increasingly utilized in industrial and clinical applications. In the present study, the method of chemical conjugation by affinity peptide (CCAP) for site-specific chemical modification has been developed by using a peptide that bound with high affinity to human IgG-Fc. This method enabled a rapid modification of a specific residue (Lys248 on Fc) in a one-step reaction under mild condition to form a stable amide bond between the peptide and Fc. The monovalent peptide-IgG conjugate not only maintained complete antigen binding but also bound to Fc receptors (FcRn, FcγRI, and FcγRIIIa), indicating that it is a suitable conjugate form that can be further developed into highly functional antibody therapeutics. CCAP was applied for the preparation of an antibody-drug conjugate and a bispecific antibody to demonstrate the usefulness of this method.

IgY-binding peptide screened from a random peptide library as a ligand for IgY purification.

Chicken egg yolk immunoglobulin (IgY) is a functional substitute for mammalian IgG for antigen detection. Traditional IgY purification methods involve multi-step procedures resulting in low purity and recovery of IgY. In this study, we developed a simple IgY purification system using IgY-specific peptides identified by T7 phage display technology. From disulfide-constrained random peptide libraries constructed on a T7 phage, we identified three specific binding clones (Y4-4, Y5-14, and Y5-55) through repeated biopanning. The synthetic peptides showed high binding specificity to IgY-Fc and moderate affinity for IgY-Fc (Kd : Y4-4 = 7.3 ± 0.2 µM and Y5-55 = 4.4 ± 0.1 µM) by surface plasmon resonance analysis. To evaluate the ability to purify IgY, we performed immunoprecipitation and affinity high-performance liquid chromatography using IgY-binding peptides; the result indicated that these peptides can be used as affinity ligands for IgY purification. We then used a peptide-conjugated column to purify IgY from egg yolks pre-treated using an optimized delipidation technique. Here, we report the construction of a cost-effective, one-step IgY purification system, with high purity and recovery. © 2017 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

Pharmacophore generation from a drug-like core molecule surrounded by a library peptide via the 10BASEd-T on bacteriophage T7.

We have achieved site-specific conjugation of several haloacetamide derivatives into designated cysteines on bacteriophage T7-displayed peptides, which are fused to T7 capsid protein gp10. This easiest gp10 based-thioetherification (10BASEd-T) undergoes almost quantitatively like a click reaction without side reaction or loss of phage infectivity. The post-translational modification yield, as well as the site-specificity, is quantitatively analyzed by a fluorescent densitometric analysis after gel electrophoresis. The detailed structure of the modified peptide on phage is identified with tandem mass spectrometry. Construction of such a peptide-fused phage library possessing non-natural core structures will be useful for future drug discovery. For this aim, we propose a novel concept of pharmacophore generation from a drug-like molecule (i.e., salicylic acid) conjugated with surrounding randomized peptides. By using the hybrid library, streptavidin-specific binders are isolated through four rounds of biopanning.

Human IgA-binding peptides selected from random peptide libraries: affinity maturation and application in IgA purification.

Phage display system is a powerful tool to design specific ligands for target molecules. Here, we used disulfide-constrained random peptide libraries constructed with the T7 phage display system to isolate peptides specific to human IgA. The binding clones (A1-A4) isolated by biopanning exhibited clear specificity to human IgA, but the synthetic peptide derived from the A2 clone exhibited a low specificity/affinity (K(d) = 1.3 µm). Therefore, we tried to improve the peptide using a partial randomized phage display library and mutational studies on the synthetic peptides. The designed Opt-1 peptide exhibited a 39-fold higher affinity (K(d) = 33 nm) than the A2 peptide. An Opt-1 peptide-conjugated column was used to purify IgA from human plasma. However, the recovered IgA fraction was contaminated with other proteins, indicating nonspecific binding. To design a peptide with increased binding specificity, we examined the structural features of Opt-1 and the Opt-1-IgA complex using all-atom molecular dynamics simulations with explicit water. The simulation results revealed that the Opt-1 peptide displayed partial helicity in the N-terminal region and possessed a hydrophobic cluster that played a significant role in tight binding with IgA-Fc. However, these hydrophobic residues of Opt-1 may contribute to nonspecific binding with other proteins. To increase binding specificity, we introduced several mutations in the hydrophobic residues of Opt-1. The resultant Opt-3 peptide exhibited high specificity and high binding affinity for IgA, leading to successful isolation of IgA without contamination.

Reduction of the Cytotoxicity of Copper (II) Oxide Nanoparticles by Coating with a Surface-Binding Peptide.

Copper (II) oxide nanoparticles (CuO-NPs) have been studied as potential antimicrobial agents, similar to silver or platinum nanoparticles. However, the use of excess NPs is limited by their safety and toxicity in beneficial microflora and human cells. In this study, we evaluated the cytotoxicity of CuO-NPs by coating with a novel cyclic peptide, CuO binding peptide 1 (CuBP1), cyclic-SCATPFSPQVCS, which binds to the surface of CuO-NPs. CuBP1 was identified using biopanning of a T7 phage display system and was found to promote the aggregation of CuO-NPs under mild conditions. The treated CuO-NPs with CuBP1 caused the reduction of the cytotoxicity against Escherichia coli, Lactobacillus helveticus, and five other microorganisms, including bacteria and eukaryotes. Similar effects were also demonstrated against human embryonic kidney (HEK293) cells in vitro. Our findings suggested that the CuO-NPs coated with a surface-binding peptide may have applications as a safe antimicrobial agent without excessive cytotoxic activity against beneficial microflora and human cells. Moreover, a similar tendency may be achieved with other metal particles, such as silver or platinum NPs, by using optimal metal binding peptides.

The origins of binding specificity of a lanthanide ion binding peptide.

Lanthanide ions (Ln3+) show similar physicochemical properties in aqueous solutions, wherein they exist as + 3 cations and exhibit ionic radii differences of less than 0.26 Å. A flexible linear peptide lanthanide binding tag (LBT), which recognizes a series of 15 Ln3+, shows an interesting characteristic in binding specificity, i.e., binding affinity biphasically changes with an increase in the atomic number, and shows a greater than 60-fold affinity difference between the highest and lowest values. Herein, by combining experimental and computational investigations, we gain deep insight into the reaction mechanism underlying the specificity of LBT3, an LBT mutant, toward Ln3+. Our results clearly show that LBT3-Ln3+ binding can be divided into three, and the large affinity difference is based on the ability of Ln3+ in a complex to be directly coordinated with a water molecule. When the LBT3 recognizes a Ln3+ with a larger ionic radius (La3+ to Sm3+), a water molecule can interact with Ln3+ directly. This extra water molecule infiltrates the complex and induces dissociation of the Asn5 sidechain (one of the coordinates) from Ln3+, resulting in a destabilizing complex and low affinity. Conversely, with recognition of smaller Ln3+ (Sm3+ to Yb3+), the LBT3 completely surrounds the ions and constructs a stable high affinity complex. Moreover, when the LBT3 recognizes the smallest Ln3+, namely Lu3+, although it completely surrounds Lu3+, an entropically unfavorable phenomenon specifically occurs, resulting in lower affinity than that of Yb3+. Our findings will be useful for the design of molecules that enable the distinction of sub-angstrom size differences.

Expression of the extracellular region of the human interleukin-4 receptor alpha chain and interleukin-13 receptor alpha1 chain by a silkworm-baculovirus system.

The receptor binding to interleukin (IL)-13 is composed of the IL-13 receptor alpha1 chain (IL-13Ralpha1) and the IL-4 receptor alpha chain (IL-4Ralpha). In order to investigate the interaction of IL-13 with IL-13Ralpha1 and IL-4Ralpha, the DNA fragments coding the extracellular regions of human IL-13Ralpha1 and the IL-4Ralpha (containing a cytokine receptor homologous region) were fused with mouse Fc and expressed by a silkworm-baculovirus system. The expressed receptors were successfully purified by affinity chromatography using protein A, and the Fc region was removed by thrombin digestion. After further purification with anion-exchange chromatography, these receptors were used to investigate the ligand-receptor interaction. Size exclusion chromatography and SPR analysis revealed that mixture of IL-13 and IL-13Ralpha1 showed predominant affinity to IL-4Ralpha, although neither detectable affinity of IL-13 nor IL-13Ralpha1 was observed against IL-4Ralpha. Combining these data with the moderate affinity of IL-13 to IL-13Ralpha1, this indicates that IL-13 first binds to IL-13Ralpha1 and recruits consequently to IL-4R.

Ordered silica mineralization by regulating local reaction conditions.

Using cationic peptides with tetramethyl orthosilicate, a silica nano-film >100 µm in size with <100 nm thickness was constructed under physiological conditions. Control of silica nucleation speed and location was found to be the dominant factor affecting the ordered architecture. Our approach adds new insight into bottom-up nanomaterial construction and contributes to evaluating the silica mineralization system in living organisms.

Rationally designed mineralization for selective recovery of the rare earth elements.

The increasing demand for rare earth (RE) elements in advanced materials for permanent magnets, rechargeable batteries, catalysts and lamp phosphors necessitates environmentally friendly approaches for their recovery and separation. Here, we propose a mineralization concept for direct extraction of RE ions with Lamp (lanthanide ion mineralization peptide). In aqueous solution containing various metal ions, Lamp promotes the generation of RE hydroxide species with which it binds to form hydrophobic complexes that accumulate spontaneously as insoluble precipitates, even under physiological conditions (pH ∼6.0). This concept for stabilization of an insoluble lanthanide hydroxide complex with an artificial peptide also works in combination with stable scaffolds like synthetic macromolecules and proteins. Our strategy opens the possibility for selective separation of target metal elements from seawater and industrial wastewater under mild conditions without additional energy input.

Construction of a crown ether-like supramolecular library by conjugation of genetically-encoded peptide linkers displayed on bacteriophage T7.

By using the 10BASEd-T, we have synthesized a crown ether-like macrocyclic library possessing randomized peptide linkers on bacteriophage T7. Among 1.5 × 10(9) diversities of the supramolecule candidates, we have obtained a specific binder for the N-terminal domain of Hsp90.

Gp10 based-thioetherification (10BASE(d)-T) on a displaying library peptide of bacteriophage T7.

The site-specific introduction of a haloacetamide derivative into a designated cysteine on a displaying peptide on a capsid protein (gp10) of bacteriophage T7 has been achieved. This easiest gp10-based thioetherification (10BASEd-T) is carried out in one-pot without side reactions or loss of phage infectivity.

Regulation of T cell response by blocking the ICOS signal with the B7RP-1-specific small antibody fragment isolated from human antibody phage library.

A costimulatory signal is required for the full activation of T cells, in addition to the antigen-specific signal via the T cell receptor. The inducible costimulator, ICOS is one of the costimulatory molecules that play an essential role in this process, particularly in the expansion or the development of effector T cells. As blocking of the interaction between ICOS and its ligand, B7RP-1, suppresses the T cell response, it can be applied to the treatment of allograft rejection or autoimmune diseases. Here, we isolated four scFv clones that were specific to human B7RP-1 by biopanning a human antibody phage library. We found that three of these clones inhibited the interaction between ICOS-Fc and B7RP-1-Fc. These inhibitory clones not only recognized B7RP-1 molecules expressed on B cells, as assessed by FACS, but also exhibited inhibitory activity in a proliferation assay of T cells stimulated with anti-CD3 mAb and B7RP-1-Fc. Finally, the suppression effect of the scFv on the allogenic immune response was examined using a mixed lymphocyte reaction assay, which demonstrated a successful inhibition of the allogenic reaction, in spite of the high dose needed for complete inhibition (360 nM).

Discovery and characterization of a peptide motif that specifically recognizes a non-native conformation of human IgG induced by acidic pH conditions.

In therapeutic antibody preparation, acidic pH conditions are generally used for elution from Protein A affinity column of IgG or for its viral inactivation. Exposing IgG to low pH conditions induces conformational changes, leading to its functional damage or loss, although the mechanisms have not been fully elucidated. In this study using random peptide T7 phage display libraries, we isolated a unique and novel peptide motif that specifically recognized the non-native conformer (acid conformer) of human IgG that was generated by the low pH treatment, but not the native conformer. We examined the generation conditions and biochemical properties of acid conformer using the peptide motif as an affinity ligand. The acid conformer was easily generated at acidic pH (25 degrees C). The peptides isolated here could contribute to the elucidation of the mechanisms of antibody dysfunction or aggregation during acid exposure as well as storage of human IgG.