Selection of turning-on fluorogenic probe as protein-specific detector obtained via the 10BASEd-T

Abstract

ARTICLES YOU MAY BE INTERESTED INAbstract. In order to obtain a molecular probe for specific protein detection, we have synthesized fluorogenic probe library of vast diversity on bacteriophage T7 via the gp10 based-thioetherification (10BASEd-T). A remarkable turningon probe which is excitable by widely applicable visible light was selected from the library.

Combinatorially Screened Peptide as Targeted Covalent Binder: Alteration of Bait-Conjugated Peptide to Reactive Modifier

“…As a model target protein, we chose glutathione S-transferase (GST) because rationally designed covalent GST binders in which a warhead is conjugated with artificial reporter tags or a natural ligand (i.e., glutathione) have been reported; their structure information as well as conjugation efficiency upon GST binding could be easily compared with our combinatorially screened binder. For the bait fragments with different shapes, we have chosen several small or middle-sized solvatochromic fluorophores with neutral charge, , so that we would sense when the bait could be buried deeply into a pocket of the target protein through hydrophobic interaction (Figure ). These fragments were independently reacted with a designated cysteine on a T7-displayed randomized peptide library, and three rounds of biopanning were performed against biotinylated-GST .…”

“…For the bait fragments with different shapes, we have chosen several small or middle-sized solvatochromic fluorophores with neutral charge, , so that we would sense when the bait could be buried deeply into a pocket of the target protein through hydrophobic interaction (Figure ). These fragments were independently reacted with a designated cysteine on a T7-displayed randomized peptide library, and three rounds of biopanning were performed against biotinylated-GST . After the selection process, amino acid sequences of the polyclonal binders possessing each fragment were directly analyzed by a next generation sequencer (NGS).…”

“…Synthesis of each bait-conjugated peptide library on phage and biopanning against GST was performed as described previously . In brief, approximately 1.0 × 10 11 pfu of T7Select10 library (-S-G-G-G-X 3 -C-X 5–7 -C-X 3 ; X represents any randomized amino acid) was modified with bromoacetamide (BA)-conjugated bait fragment via the 10BASE d -T. After modification, the T7 phage library was dissolved in selection buffer (PBS supplemented with 0.1% v/v TritonX-100).…”

“…To avoid the latter problem of unnecessary side reactions, here we take a detoured selection strategy to obtain a peptide-type covalent binder possessing target specificity (Figure ). First, we introduce several solvatochromic bait fragments, instead of the warhead, to designated cysteine on T7 phage-displayed library peptides via the gp10-based thioetherification (10BASE d -T). , Second, we obtain targeted non -covalent binders from each bait-conjugated peptide library. Third, when a consensus peptide sequence around the designated cysteine appears, no matter what kind of bait fragment is used, we alter the bait in the consensus peptide into a differently structured warhead, to obtain the targeted covalent binder.…”

“…To avoid the latter problem of unnecessary side reactions, here we take a detoured selection strategy to obtain a peptidetype covalent binder possessing target specificity (Figure 1). First, we introduce several solvatochromic bait fragments, instead of the warhead, to designated cysteine on T7 phagedisplayed library peptides 22 via the gp10-based thioetherification (10BASE d -T). 23,24 Second, we obtain targeted noncovalent binders from each bait-conjugated peptide library.…”