The Design and Application of Inhibiting Peptide for Rapid In vitro Downregulation of Post-Translational Modifications Levels at a Specific Site.

Proteins are the primary executors of life activities and are regulated at transcriptional, translational, and post-translational modification (PTM) levels. Moreover, PTM represents a more complicated mechanism for regulating protein activities as a protein generally has various types of PTMs and multiple sites for a specific PTM. Plasmid transfection or lentivirus and adenovirus could be used in initial screening of the functionally important PTM site among all identified sites; however, these methods always face challenges such as low-efficiency in cell and tissue entry, time-consuming and high costs, potential immune reaction, etc. To address this, we recently developed and successfully employed a type of recombined peptide called TAT-PIP, TAT-conjugated PTM inhibitory peptide. TAT-PIP consists of a TAT module that facilitates cell and tissue entry and a PIP module that specifically downregulates the PTM at targeted sites through competitive binding. Here, we present a protocol that outlines the design specifications, quality control standards, and functional verification system of TAT-PIP. In the design section, we describe the consistency, the optional length, specificity, and conservation of TAT-PIP. Next, we introduce the quality testing requirements of TAT-PIP, which guarantee its efficacy and safety. In the application of the TAT-PIP part, we introduced the concentration gradient test of TAT-PIP, the incubation process of the tested cells, and the subsequent phenotypic detection. In summary, we describe an effective method for screening PTM sites by selectively knocking down a specific site and observing the resulting phenotype to infer its function. Due to its low synthetic cost and high efficiency, this method overcomes the limitations of existing technologies, such as plasmid transfection.