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In Vitro Selection of Cathepsin E-Activity-Enhancing Peptide Aptamers at Neutral pH.

Biyani M, Futakami M, Kitamura K, Kawakubo T, Suzuki M, Yamamoto K, Nishigaki K - Int J Pept (2011)

Bottom Line: 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.This method is expected to be widely applicable for the identification of protease-activity-enhancing peptide aptamers.

View Article: PubMed Central - PubMed

Affiliation: Department of Functional Materials Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Saitama 338-8570, Japan.

ABSTRACT
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 (K(D); ∼300 nM), 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.

No MeSH data available.


Related in: MedlinePlus

Surveying the building block peptides used for the secondary library. (a) Cluster analysis of the peptides selected from the primary library, using ClustalW supported by DDBJ (DNA Data Bank of Japan). Consensus sequences are shown in bold. (b) Schematic drawing of 23 tetramer peptide blocks used for the secondary library construction by the ASAC (all-steps-all-combinations) method. In addition to the blocks from the selected sequences (which also contain the SF-link construct), arbitrarily chosen blocks of (GGGG)/n were combined.
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fig2: Surveying the building block peptides used for the secondary library. (a) Cluster analysis of the peptides selected from the primary library, using ClustalW supported by DDBJ (DNA Data Bank of Japan). Consensus sequences are shown in bold. (b) Schematic drawing of 23 tetramer peptide blocks used for the secondary library construction by the ASAC (all-steps-all-combinations) method. In addition to the blocks from the selected sequences (which also contain the SF-link construct), arbitrarily chosen blocks of (GGGG)/n were combined.

Mentions: The primary and the secondary libraries were constructed according to the protocols described in [9]. In brief, the primary library was constructed by Y-ligation-based block shuffling (YLBS) [19]. The secondary library was constructed by exploiting the YLBS method with slight modifications. The peptide sequences obtained from the primary library selection were cluster-analyzed and used to design the subsequently constructed blocks (Figure 2). Using these blocks, YLBS-shuffling was performed (Table 1). The resultant library contained all of the arbitrarily shuffled blocks with a different number of blocks (1–4 blocks; Figure 2(b)), thus termed an all-steps-all-combinations (ASAC) library. Prior to the in vitro selection, the variable sequences were integrated into the cDNA-tagged-SF-link peptide construct. The preparation of the construct was performed following the method previously reported [9] and is partially appearing in Figure 1.


In Vitro Selection of Cathepsin E-Activity-Enhancing Peptide Aptamers at Neutral pH.

Biyani M, Futakami M, Kitamura K, Kawakubo T, Suzuki M, Yamamoto K, Nishigaki K - Int J Pept (2011)

Surveying the building block peptides used for the secondary library. (a) Cluster analysis of the peptides selected from the primary library, using ClustalW supported by DDBJ (DNA Data Bank of Japan). Consensus sequences are shown in bold. (b) Schematic drawing of 23 tetramer peptide blocks used for the secondary library construction by the ASAC (all-steps-all-combinations) method. In addition to the blocks from the selected sequences (which also contain the SF-link construct), arbitrarily chosen blocks of (GGGG)/n were combined.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3064998&req=5

fig2: Surveying the building block peptides used for the secondary library. (a) Cluster analysis of the peptides selected from the primary library, using ClustalW supported by DDBJ (DNA Data Bank of Japan). Consensus sequences are shown in bold. (b) Schematic drawing of 23 tetramer peptide blocks used for the secondary library construction by the ASAC (all-steps-all-combinations) method. In addition to the blocks from the selected sequences (which also contain the SF-link construct), arbitrarily chosen blocks of (GGGG)/n were combined.
Mentions: The primary and the secondary libraries were constructed according to the protocols described in [9]. In brief, the primary library was constructed by Y-ligation-based block shuffling (YLBS) [19]. The secondary library was constructed by exploiting the YLBS method with slight modifications. The peptide sequences obtained from the primary library selection were cluster-analyzed and used to design the subsequently constructed blocks (Figure 2). Using these blocks, YLBS-shuffling was performed (Table 1). The resultant library contained all of the arbitrarily shuffled blocks with a different number of blocks (1–4 blocks; Figure 2(b)), thus termed an all-steps-all-combinations (ASAC) library. Prior to the in vitro selection, the variable sequences were integrated into the cDNA-tagged-SF-link peptide construct. The preparation of the construct was performed following the method previously reported [9] and is partially appearing in Figure 1.

Bottom Line: 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.This method is expected to be widely applicable for the identification of protease-activity-enhancing peptide aptamers.

View Article: PubMed Central - PubMed

Affiliation: Department of Functional Materials Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Saitama 338-8570, Japan.

ABSTRACT
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 (K(D); ∼300 nM), 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.

No MeSH data available.


Related in: MedlinePlus