Abstract
We have achieved site-specific conjugation of several haloacetamide derivatives into designated cysteines on bacteriophage T7-displayed peptides, which are fused to T7 capsid protein gp10. This easiest gp10 based-thioetherification (10BASEd-T) undergoes almost quantitatively like a click reaction without side reaction or loss of phage infectivity. The post-translational modification yield, as well as the site-specificity, is quantitatively analyzed by a fluorescent densitometric analysis after gel electrophoresis. The detailed structure of the modified peptide on phage is identified with tandem mass spectrometry. Construction of such a peptide-fused phage library possessing non-natural core structures will be useful for future drug discovery. For this aim, we propose a novel concept of pharmacophore generation from a drug-like molecule (i.e., salicylic acid) conjugated with surrounding randomized peptides. By using the hybrid library, streptavidin-specific binders are isolated through four rounds of biopanning.
Highlights
Post-translational chemical modification of bacteriophage-displayed peptides is attracting attention for drug discovery [1,2,3]
Under the optimized modification conditions, we examined the infectivity of the salicylic acid (Sal)-conjugated T7 phage peptide library by plaque assay and found that the modified T7 phage retained its infectivity (Figure 3)
The structure of the modified peptide on phage was identified by tandem mass spectrometry, and the conjugation yield was estimated by SDS-PAGE followed by fluorescence imaging in a rapid and universal manner
Summary
Post-translational chemical modification of bacteriophage (referred to hereafter as phage). Multiple research groups have reported construction of non-natural peptide libraries by the post-translational chemical modifications of M13 phage-displayed peptides [5,6,7,8,9,10]. We have constructed a non-natural peptide library by the post-translational chemical modification of T7 phage-displayed peptides, namely gp based-thioetherification (10BASEd-T) [14]. 4-iodoacetamidosalicylic acid (Sal-IA) was used because it is one of the smallest pharmaceutical molecules possessing both hydrophobic benzene ring and hydrophilic hydroxyl/carboxyl groups. These groups may potentially interact with various proteins through hydrophobic/π-interactions and hydrogen bonding [15], and seldom form aggregates [16]. We constructed an artificial library of salicylic acid (Sal) possessing randomized peptides via the 10BASEd-T
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