Angiotensin I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata.

We examined the inhibitory activity of angiotensin I converting enzyme (ACE) in protein hydrolysates from dulse, Palmaria palmata. The proteins extracted from dulse were mainly composed of phycoerythrin (PE) followed by phycocyanin (PC) and allophycocyanin (APC). The dulse proteins showed slight ACE inhibitory activity, whereas the inhibitory activity was extremely enhanced by thermolysin hydrolysis. The ACE inhibitory activity of hydrolysates was hardly affected by additional pepsin, trypsin and chymotrypsin treatments. Nine ACE inhibitory peptides (YRD, AGGEY, VYRT, VDHY, IKGHY, LKNPG, LDY, LRY, FEQDWAS) were isolated from the hydrolysates by reversed-phase high-performance liquid chromatography (HPLC), and it was demonstrated that the synthetic peptide LRY (IC50: 0.044 µmol) has remarkably high ACE inhibitory activity. Then, we investigated the structural properties of dulse phycobiliproteins to discuss the origin of dulse ACE inhibitory peptides. Each dulse phycobiliprotein possesses α-subunit (Mw: 17,477-17,638) and ß-subunit (Mw: 17,455-18,407). The sequences of YRD, AGGEY, VYRT, VDHY, LKNPG and LDY were detected in the primary structure of PE α-subunit, and the LDY also exists in the APC α- and ß-subunits. In addition, the LRY sequence was found in the ß-subunits of PE, PC and APC. From these results, it was suggested that the dulse ACE inhibitory peptides were derived from phycobiliproteins, especially PE. To make sure the deduction, we carried out additional experiment by using recombinant PE. We expressed the recombinant α- and ß-subunits of PE (rPEα and rPEß, respectively), and then prepared their peptides by thermolysin hydrolysis. As a result, these peptides showed high ACE inhibitory activities (rPEα: 94.4%; rPEß: 87.0%). Therefore, we concluded that the original proteins of dulse ACE inhibitory peptides were phycobiliproteins.

Establishment of a reborn MMV-microarray technology: realization of microbiome analysis and other hitherto inaccessible technologies.

BACKGROUND: With the accelerating development of bioscience, the problem of research cost has become important. We previously devised and developed a novel concept microarray with manageable volumes (MMV) using a soft gel. It demonstrated the great potential of the MMV technology with the examples of 1024-parallel-cell culture and PCR experiments. However, its full potential failed to be expressed, owing to the nature of the material used for the MMV chip. RESULTS: In the present study, by developing plastic-based MMVs and associated technologies, we introduced novel technologies such as C2D2P (in which the cells in each well are converted from DNA to protein in 1024-parallel), NGS-non-dependent microbiome analysis, and other powerful applications. CONCLUSIONS: The reborn MMV-microarray technology has proven to be highly efficient and cost-effective (with approximately 100-fold cost reduction) and enables us to realize hitherto unattainable technologies.

Novel concept microarray enabling PCR and multistep reactions through pipette-free aperture-to-aperture parallel transfer.

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 Aß-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.

Development of systemic in vitro evolution and its application to generation of peptide-aptamer-based inhibitors of cathepsin E.

Proteases are involved in various biological functions. Thus, inhibition of their activities is scientifically interesting and medically important. However, there is no systematic method established to date to generate endopeptidase inhibitory peptides. Here, we report a general system to identify endopeptidase inhibitory peptides based on the use of in vitro evolution. Using this system, we generated peptides that inhibit cathepsin E (CE) specifically at a submicromolar IC(50). This system generates protease inhibitor peptides utilizing techniques of cDNA display, selection-by-function, Y-ligation-based block shuffling, and others. We further demonstrated the importance and effectiveness of a secondary library for obtaining small-sized and active peptides. CE inhibitory peptides generated by this method were characterized by a small size (8 to 12 aa) and quite different sequences, suggesting that they bind to different sites on CE. Typical CE inhibitory peptide aptamers obtained here (P(i)101; SCGG IIII SCIA) have half an inhibition activity (K(i); 5 nM) of pepstatin A (potent CE inhibitor) without inhibiting cathepsin D (structurally similar to CE). The general applicability of this system suggests that it may be useful to identify inhibitory peptides for various kinds of proteases and that it may therefore contribute to protein science and drug discovery. The peptide binding to a protein is discussed in comparison with the antibody binding to an antigen.

Selection-by-function: efficient enrichment of cathepsin E inhibitors from a DNA library.

A method for efficient enrichment of protease inhibitors out of a DNA library was developed by introducing SF-link technology. A two-step selection strategy was designed consisting of the initial enrichment of aptamers based on binding function while the second enrichment step was based on the inhibitory activity to a protease, cathepsin E (CE). The latter was constructed by covalently linking of a biotinylated peptide substrate to each of the ssDNA molecule contained in the preliminarily selected DNA library, generating 'SF-link'. Gradual enrichment of inhibitory DNAs was attained in the course of selection. One molecule, SFR-6-3, showed an IC(50) of around 30 nM, a K(d) of around 15 nM and high selectivity for CE. Sequence and structure analysis revealed a C-rich sequence without any guanine and possibly an i-motif structure, which must be novel to be found in in vitro-selected aptamers. SF-link technology, which is novel as the screening technology, provided a remarkable enrichment of specific protease inhibitors and has a potential to be further developed.

Construction of block-shuffled libraries of DNA for evolutionary protein engineering: Y-ligation-based block shuffling.

Evolutionary protein engineering is now proceeding to a new stage in which novel technologies, besides the conventional point mutations, to generate a library of proteins, are required. In this context, a novel method for shuffling and rearranging DNA blocks (leading to protein libraries) is reported. A cycle of processes for producing combinatorial diversity was devised and designated Y-ligation-based block shuffling (YLBS). Methodological refinement was made by applying it to the shuffling of module-sized and amino acid-sized blocks. Running three cycles of YLBS with module-sized GFP blocks resulted in a high diversity of an eight-block shuffled library. Partial shuffling of the central four blocks of GFP was performed to obtain in-effect shuffled protein, resulting in an intact arrangement. Shuffling of amino acid monomer-sized blocks by YLBS was also performed and a diversity of more than 10(10) shuffled molecules was attained. The deletion problems encountered during these experiments were shown to be solved by additional measures which tame type IIS restriction enzymes. The frequency of appearance of each block was skewed but was within a permissible range. Therefore, YLBS is the first general method for generating a huge diversity of shuffled proteins, recombining domains, exons and modules with ease.