A novel agonist with homobivalent single-domain antibodies that bind the FGF receptor 1 domain III functions as an FGF2 ligand.

Fibroblast growth factor (FGF) is a multifunctional protein that exhibits a wide range of biological effects. Most commonly, it acts as a mitogen, but it also has regulatory, morphological, and endocrine effects. The four receptor subtypes of FGF are activated by more than 20 different FGF ligands. FGF2, one of the FGF ligands, is an essential factor for cell culture in stem cells for regenerative medicine; however, recombinant FGF2 is extremely unstable. Here, we successfully generated homobivalent agonistic single-domain antibodies (variable domain of heavy chain of heavy chain antibodies referred to as VHHs) that bind to domain III and induce activation of the FGF receptor 1 and thus transduce intracellular signaling. This agonistic VHH has similar biological activity (EC50) as the natural FGF2 ligand. Furthermore, we determined that the agonistic VHH could support the proliferation of human-induced pluripotent stem cells (PSCs) and human mesenchymal stem cells, which are PSCs for regenerative medicine. In addition, the agonistic VHH could maintain the ability of mesenchymal stem cells to differentiate into adipocytes or osteocytes, indicating that it could maintain the properties of PSCs. These results suggest that the VHH agonist may function as an FGF2 mimetic in cell preparation of stem cells for regenerative medicine with better cost effectiveness.

In vitro selection of anti-gliadin single-domain antibodies from a naïve library for cDNA-display mediated immuno-PCR.

Gluten intolerance, or adverse intestinal reactions to gluten, is a fairly common problem among certain groups of people. Celiac disease is the most severe form of gluten intolerance, which can lead to permanent damage in the digestive system. Since lifelong avoidance of gluten is the only available treatment, development of reliable techniques to identify gluten contamination in food is important. Gliadin, a component of gluten, is known to play a major role in gluten toxicity. In this study, cDNA display method was used to select specific single-domain antibodies against toxic gliadin from an alpaca-derived naïve VHH library. The cDNA display method is a promising in vitro display technique, which uniquely converts an unstable mRNA-protein fusion molecule to a stable mRNA/cDNA-protein fusion molecule using a well-designed puromycin linker. Three candidate VHHs were selected and the affinities of the VHHs were observed by pulldown assay and indirect ELISA method. In addition, a novel cDNA display mediated immuno-PCR method (cD-IPCR) was successfully applied to detect gliadin in food. We believe this work demonstrates the potential application of the cDNA display method in selecting binders against toxic and heterogeneous targets such as gliadin with an immunization-free preparation manner.

Versatile C-terminal specific biotinylation of proteins using both a puromycin-linker and a cell-free translation system for studying high-throughput protein-molecule interactions.

Immobilization of a protein in a functionally active form and correct orientation for high-throughput analysis is crucial for surface-based protein-molecular interaction studies and should aid progress in associated nanotechnologies. Here, we present a general method for controlled and oriented immobilization of proteins by a puromycin-linker for cDNA display technology. The utility and potential of this method was demonstrated by examining the interaction between the B domain of protein A and immunoglobulin G (IgG) by surface plasmon resonance. This study revealed that the mRNA fragment of the mRNA-protein fusion (i.e., mRNA display) interferes with the interaction between the protein (B domain) and its target molecule (IgG). This results in a reduction of the apparent affinity by ~10-fold. This method is expected to find wide appeal in the fields of surface-based studies of protein-protein interactions, drug screening, and single molecule analysis that require only a small amount of protein sample.

Increasing the library size in cDNA display by optimizing purification procedures.

BACKGROUND: The library size is critical for selection in evolutionary molecular engineering (directed evolution). Although cDNA display has become a promising in vitro display technology by overcoming the instability of mRNA display, it is hindered by low yields. In this study, we improved the yield of cDNA display molecules by carefully examining each step of the preparation process. FINDINGS: We found that steric hindrance of ribosomes binding to the mRNA-protein fusion molecules was interfering with biotin-streptavidin binding. Additionally, reducing buffer exchange by performing RNase digestion in the His-tag-binding buffer to release the cDNA display molecules improved their His-tag purification. CONCLUSION: Our optimized conditions have improved the yield of cDNA display molecules by more than 10 times over currently used methods, making cDNA display more practically available in evolutionary molecular engineering.

Construction of a Humanized Artificial VHH Library Reproducing Structural Features of Camelid VHHs for Therapeutics.

A variable domain of heavy chain antibody (VHH) has different binding properties than conventional antibodies. Conventional antibodies prefer binding to the convex portion of the antigen, whereas VHHs prefer epitopes, such as crevices and clefts on the antigen. Therefore, developing candidates with the binding characteristics of camelid VHHs is important. Thus, To this end, a synthetic VHH library that reproduces the structural properties of camelid VHHs was constructed. First, the characteristics of VHHs were classified according to the paratope formation based on crystal structure analyses of the complex structures of VHHs and antigens. Then, we classified 330 complementarity-determining region 3 (CDR3) structures of VHHs from the Protein Data Bank (PDB) into three loop structures: Upright, Half-Roll, and Roll. Moreover, these structures depended on the number of amino acid residues within CDR3. Furthermore, in the Upright loops, several amino acid residues in the FR2 are involved in the paratope formation, along with CDR3, suggesting that the FR2 design in the synthetic library is important. A humanized synthetic VHH library, comprising two sub-libraries, Upright and Roll, was constructed and named PharmaLogical. A validation study confirmed that our PharmaLogical library reproduces VHHs with the characteristics of the paratope formation of the camelid VHHs, and shows good performance in VHH screening.

Interleukin-17A Peptide Aptamers with an Unexpected Binding Moiety Selected by cDNA Display under Heterogenous Conditions.

Peptide-based drugs are an attractive new modality of therapeutics, and in vitro selection from a large-scale library is a powerful way to identify new lead sequences. In conventional screenings, peptide specificity and stability in physiological heterogenous environments are not evaluated, which sometimes makes subsequent optimization difficult. Here we show that selection using a cDNA display system can be performed in a high percentage of serum and that this might be an option to select molecules with high potency and stability in a biological context. Specifically, we chose interleukin-17A as a target protein and performed in vitro selection of cyclic peptide aptamers from a library of approximately 1012 members in the presence of serum. The selected molecules had nanomolar affinity to the target and were stable in serum. Interestingly, we found that a component of the DNA linker that connected the peptide and cDNA may play a pivotal role in target binding.

In Vitro Construction of Large-scale DNA Libraries from Fragments Containing Random Regions using Deoxyinosine-containing Oligonucleotides and Endonuclease V.

Efficient and precise construction of DNA libraries is a fundamental starting point for directed evolution of polypeptides. Recently, several in vitro selection methods have been reported that do not rely on cells for protein expression, where peptide libraries in the order of 1013 species are used for in vitro affinity selection. To maximize their potential, simple yet versatile construction of DNA libraries from several fragments containing random regions without bacterial transformation is essential. To address this issue, we herein propose a novel DNA construction methodology based on the use of polymerase chain reaction (PCR) primers containing a single deoxyinosine (I) residue near their 5' end. Treatment of the PCR products with endonuclease V generates 3' overhangs with customized lengths and sequences, which can be ligated accurately and efficiently with other fragments having exactly complementary overhangs. As a proof of concept, we constructed an artificial gene library of single-domain antibodies from four DNA fragments.

cDNA Display: A Stable and Simple Genotype-Phenotype Coupling Using a Cell-Free Translation System.

A cDNA display method was developed based on the mRNA display method to increase its stability and efficiency for the directed evolution of various kinds of peptides and proteins. In this method, the puromycin-linker is a key molecule to realize smart genotype-phenotype coupling. A recently improved puromycin-linker and its use were explained in detail for the in vitro selection of peptides and proteins using the cDNA display method.

In Vitro Selection of Single-Domain Antibody (VHH) Using cDNA Display.

Single-domain antibody (e.g., Nanobody, VHH antibody) is a promising scaffold for therapeutic and diagnostic reagents. To expand the range of target molecules, in vitro selection using cell-free display technologies such as cDNA display is useful and powerful because of their huge libraries and robust stability. We provide technical details for in vitro selection of single-domain antibodies using cDNA display.

Enhanced mRNA-protein fusion efficiency of a single-domain antibody by selection of mRNA display with additional random sequences in the terminal translated regions.

In vitro display technologies such as mRNA and cDNA display are powerful tools to create and select functional peptides. However, in some cases, efficiency of mRNA-protein fusion is very low, which results in decreased library size and lower chance of successful selection. In this study, to improve mRNA-protein fusion efficiency, we prepared an mRNA display library of a protein with random N- and C-terminal coding regions consisting of 12 nucleotides (i.e. four amino acids), and performed an electrophoresis mobility shift assay (EMSA)-based selection of successfully formed mRNA display molecules. A single-domain antibody (Nanobody, or VHH) was used as a model protein, and as a result, a pair of sequences was identified that increased mRNA-protein fusion efficiency of this protein by approximately 20%. Interestingly, enhancement of the fusion efficiency induced by the identified sequences was protein-specific, and different results were obtained for other proteins including VHHs with different CDRs. The results suggested that conformation of mRNA as a whole, rather than the amino acid sequence of the translated peptide, is an important factor to determine mRNA-protein fusion efficiency.

An RNA Binding Peptide Consisting of Four Types of Amino Acid by in Vitro Selection Using cDNA Display.

RNA-protein interactions have a central role in the living world. In this article, we examined whether primitive peptides (30 residues) consisting of four types of amino acid (Gly, Ala, Asp, and Val) could interact with tRNA as a model of primitive RNAs in the RNA world. By in vitro selection of binding peptides using the cDNA display method, a characteristic peptide was selected from a random peptide library and assayed by electrophoretic mobility shift and pull-down assays. Interestingly, the selected peptide bound to a single-stranded region including a loop structure of an RNA molecule with some sequence specificity.

Easy and rapid binding assay for functional analysis of disulfide-containing peptides by a pull-down method using a puromycin-linker and a cell-free translation system.

Constrained peptides are an attractive class as affinity reagents or drug leads owing to their excellent binding properties. Many kinds of these peptides, such as cyclic peptides containing disulfide bridges, are found in nature or designed artificially by directed evolution. However, confirming the binding properties of the disulfide-rich peptides can be generally difficult, because of oxidative folding problems in the preparation steps. Therefore, a method for evaluating the binding properties of such peptides rapidly and easily is required. Here, we report an easy and rapid method for preparing biotin-attached peptides containing disulfide bridges or a chemical cross-linker using a cell-free translation system and a puromycin-linker, which is applicable to pull-down assays for protein (or peptide) molecular interaction analysis.