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Biyani M.,Saitama University | Biyani M.,Saitama Small and Medium Enterprises Development Corporation | Futakami M.,Saitama University | Futakami M.,Saitama Small and Medium Enterprises Development Corporation | And 10 more authors.
International Journal of Peptides | Year: 2011

The aspartic protease cathepsin E has been shown to induce apoptosis in cancer cells under physiological conditions. Therefore, cathepsin E-activity-enhancing peptides functioning in the physiological pH range are valuable potential cancer therapeutic candidates. Here, we have used a general in vitro selection method (evolutionary rapid panning analysis system (eRAPANSY)), based on inverse substrate-function link (SF-link) selection to successfully identify cathepsin E-activity-enhancing peptide aptamers at neutral pH. A successive enrichment of peptide activators was attained in the course of selection. One such peptide activated cathepsin E up to 260, had a high affinity (KD; 300nM), and had physiological activity as demonstrated by its apoptosis-inducing reaction in cancerous cells. This method is expected to be widely applicable for the identification of protease-activity-enhancing peptide aptamers. Copyright © 2011 Madhu Biyani et al.

Kinoshita Y.,Saitama University | Tayama T.,Saitama University | Kitamura K.,Saitama University | Kitamura K.,Janusys Corporation | And 14 more authors.
BMC Biotechnology | Year: 2010

Background: The microarray has contributed to developing the omic analysis. However, as it depends basically on the surface reaction, it is hard to perform bulk reactions and sequential multistep reactions. On the other hand, the popular microplate technology, which has a great merit of being able to perform parallel multistep reactions, has come to its limit in increasing the number of wells (currently, up to 9600) and reducing the volume to deal with due to the difficulty in operations.Results: Here, we report a novel microarray technology which enables us to explore advanced applications, termed microarray-with-manageable volumes (MMV). The technical essence is in the pipette-free direct parallel transfer from well to well performed by centrifugation, evading the evaporation and adsorption-losses during handling. By developing the MMV plate, accompanying devices and techniques, generation of multiple conditions (256 kinds) and performance of parallel multistep reactions, including PCR and in vitro translation reactions, have been made possible. These were demonstrated by applying the MMV technology to searching lysozyme-crystallizing conditions and selecting peptides aimed for Aβ-binding or cathepsin E-inhibition.Conclusions: With the introduction of a novel concept microarray (MMV) technology, parallel and multistep reactions in sub-μL scale have become possible. © 2010 Kinoshita et al; licensee BioMed Central Ltd.

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.

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