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Kawaguchi, Japan

Ueno S.,Saitama University | Kimura S.,Saitama University | Kimura S.,Janusys Corporation and 508 | Ichiki T.,University of Tokyo | Nemoto N.,Saitama University
Journal of Biotechnology | Year: 2012

cDNA display using a puromycin-linker to covalently bridge a protein and its coding cDNA is a stable and efficient in vitro protein selection method. The optimal design of the often-used puromycin-linker is vital for effective selection. In this report, an improved puromycin-linker containing deoxyinosine bases as cleavage sites, which are recognized by endonuclease V, was introduced to extend the variety of the selection targets to molecules such as RNA. The introduction of this linker enables efficient in vitro protein selection without contamination from RNase T1, which is used for the conventional linker containing ribonucleotide G bases. In addition, mRNA-protein fusion efficiency was found to not depend on the length of the flexible poly (ethylene glycol) (PEG) region of the linker. These findings will allow practical and easy-to-use in vitro protein selection by cDNA display. © 2012 Elsevier B.V. Source

Kitamura K.,Janusys Corporation and 508 | Kitamura K.,Saitama University | Kitamura K.,Enterprise Corp | Kitamura K.,Saitama small and medium Enterprises Development Corporation | And 14 more authors.
Journal of Peptide Science | Year: 2012

Improving a particular function of molecules is often more difficult than identifying such molecules ab initio. Here, a method to acquire higher affinity and/or more functional peptides was developed as a progressive library selection method. The primary library selection products were utilized to build a secondary library composed of blocks of 4 amino acids, of which selection led to peptides with increased activity. These peptides were further converted to randomly generate paired peptides. Cathepsin E-inhibitors thus obtained exhibited the highest activities and affinities (pM order). This was also the case with cathepsin E-activating peptides, proving the methodological effectiveness. The primary, secondary, and tertiary library selections can be regarded as module-finding, module-shuffling, and module-pairing, respectively, which resembles the progression of the natural evolution of proteins. The mode of peptide binding to their target proteins is discussed in analogy to antibodies and epitopes of an antigen. © 2012 European Peptide Society and John Wiley & Sons, Ltd. Source

Naimuddin M.,Japan National Institute of Advanced Industrial Science and Technology | Naimuddin M.,Janusys Corporation and 508 | Kubo T.,Japan National Institute of Advanced Industrial Science and Technology
ACS Combinatorial Science | Year: 2016

We describe a high performance platform based on cDNA display technology by developing a new modified puromycin linker-oligonucleotide. The linker consists of four major characteristics: a "ligation site" for hybridization and ligation of mRNA by T4 RNA ligase, a "puromycin arm" for covalent linkage of the protein, a "polyadenosine site" for a longer puromycin arm and purification of protein fusions (optional) using oligo-dT matrices, and a "reverse transcription site" for the formation of stable cDNA protein fusions whose cDNA is covalently linked to its encoded protein. The linker was synthesized by a novel branching strategy and provided >8-fold higher yield than previous linkers. This linker enables rapid and highly efficient ligation of mRNA (>90%) and synthesis of protein fusions (∼50-95%) in various cell-free expression systems. Overall, this new cDNA display method provides 10-200 fold higher end-usage fusions than previous methods and benefits higher diversity libraries crucial for directed protein/peptide evolution. With the increased efficiency, this system was able to reduce the time for one selection cycle to <8 h and is potentially amenable to high-throughput systems. We demonstrate the efficiency of this system for higher throughput selections of various biomolecular interactions and achieved 30-40-fold enrichment per selection cycle. Furthermore, a 4-fold higher enrichment of Flag-tag was obtained from a doped mixture compared with that of the previous cDNA display method. A three-finger protein library was evolved to isolate superior nanomolar range binding candidates for vascular endothelial growth factor. This method is expected to provide a beneficial impact to accelerated drug discovery and proteome analysis. © 2016 American Chemical Society. Source

Naimuddin M.,Japan National Institute of Advanced Industrial Science and Technology | Naimuddin M.,Chiyoda Corporation | Naimuddin M.,Janusys Corporation and 508 | Kobayashi S.,Japan National Institute of Advanced Industrial Science and Technology | And 7 more authors.
Molecular Brain | Year: 2011

Background. Directed evolution of biomolecules such as DNA, RNA and proteins containing high diversity has emerged as an effective method to obtain molecules for various purposes. In the recent past, proteins from non-immunoglobulins have attracted attention as they mimic antibodies with respect to binding potential and provide further potential advantages. In this regard, we have attempted to explore a three-finger neurotoxin protein (3F). 3F proteins are small (∼7 kDa), structurally well defined, thermally stable and resistant to proteolysis that presents them as promising candidates for directed evolution. Results. We have engineered a snake -neurotoxin that belongs to the 3F family by randomizing the residues in the loops involved in binding with acetylcholine receptors and employing cDNA display to obtain modulators of interleukin-6 receptor (IL-6R). Selected candidates were highly specific for IL-6R with dissociation constants and IC50s in the nanomolar range. Antagonists as well as agonists were identified in an IL-6 dependent cell proliferation assay. Size minimization yielded peptides of about one-third the molecular mass of the original proteins, without significant loss of activities and, additionally, lead to the identification of the loops responsible for function. Conclusions. This study shows 3F protein is amenable to introduce amino acid changes in the loops that enable preparation of a high diversity library that can be utilized to obtain ligands against macromolecules. We believe this is the first report of protein engineering to convert a neurotoxin to receptor ligands other than the parent receptor, the identification of an agonist from non-immunoglobulin proteins, the construction of peptide mimic of IL-6, and the successful size reduction of a single-chain protein. © 2011 Naimuddin et al; licensee BioMed Central Ltd. Source

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