Gp10 based-thioetherification (10BASEd-T) on a displaying library peptide of bacteriophage T7

Abstract

The site-specific introduction of a haloacetamide derivative into a designated cysteine on a displaying peptide on a capsid protein (gp10) of bacteriophage T7 has been achieved. This easiest gp10-based thioetherification (10BASEd-T) is carried out in one-pot without side reactions or loss of phage infectivity.

Selection of Color-Changing and Intensity-Increasing Fluorogenic Probe as Protein-Specific Indicator Obtained via the 10BASEd-T

“…13,14 This gp 10 based-t hioetherificaion (10BASE d -T) is carried out in a one-pot reaction without side reactions or loss of phage infectivity. Consequently, the cores can coevolve during selection for target specific binders with the phage display technology.…”

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

“…They are categorized in two different groups in 4-DMN; ZXZC*ZXDGZ and LNYC*DGW (C* and Z represent 4-DMN-conjugated Cys and hydrophobic amino acids, respectively), and in DBD; C*DZZ and ZZC*DGZ (C* represents DBD-conjugated Cys), respectively. They showed consensus peptide sequences of GZ (underlined) in the C-terminal region, which are also identical with TMR-evolved one (i.e., ZC*XDGZ; C* represents TMR-conjugated Cys) [8] and Prodan-evolved ones (i.e., NXVSCXGZ, and NPCTGZ; C* represents Prodan-conjugated Cys) [9]. Excluding the Prodan evolvers, aspartic acid (D) was also found as the consensus sequence at the next to the C-terminal penultimate residue.…”

“…4-N,N-dimethylamino-1,8-naphthalimide-bromoacetamide (4-DMN-BA) and 4-N,N-dimethylaminosulfonyl-7-(2-aminoethylamino)-2,1,3-benzoxadiazole-bromoacetamide (DBD-BA) were reacted with T7 phage-displayed randomized peptide library (-SGGG-X3-C-X5-7-C-X3; where X represents any amino acids) [8] [13] independently via the 10BASEd-T as shown in Figure 1, to make fluorogenic libraries with vast diversity (i.e., 10 9 ). As a target protein for the proof-of-concept study, we chose glutathione S-transferase (GST) because we have already evolved tetramethylrhodamine (TMR) and Prodan to GST-specific binders [8] [9].…”

“…As a target protein for the proof-of-concept study, we chose glutathione S-transferase (GST) because we have already evolved tetramethylrhodamine (TMR) and Prodan to GST-specific binders [8] [9]. By using each fluorogenic library of 4-DMN or DBD, six rounds of biopanning were performed against biotinylated-GST and enrichment of GST binders was assessed by ELISA.…”

“…In particular, those that result in a turning-on fluorescence (i.e., increase in light intensity on target recognition) are desirable because of the ease of implementation for practical use and the readily interpretable readout [7]. To obtain antibody-like specific binders with remarkable turning-on ability, we have evolved a solvatochromic fluorophore (i.e., Prodan) on the basis of a modified T7 phage display system, namely gp10 based-thioetherification (10BASEd-T) [8] [9]. A major drawback of using Prodan is that it is designed to be excited by ultraviolet light sources, which often causes high background fluorescence when crude biological samples are used as analytes.…”