Intracellular Protein Delivery by Genetically Encoded and Structurally Constrained Cell-penetrating Peptides
Author | : Kuangyu Chen (Ph. D. in chemistry) |
Publisher | : |
Total Pages | : |
Release | : 2019 |
ISBN-13 | : OCLC:1157276826 |
ISBN-10 | : |
Rating | : 4/5 ( Downloads) |
Download or read book Intracellular Protein Delivery by Genetically Encoded and Structurally Constrained Cell-penetrating Peptides written by Kuangyu Chen (Ph. D. in chemistry) and published by . This book was released on 2019 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins are valuable research tools and therapeutic agents, but their intracellular applications are limited by poor cell-permeability. Cell penetrating peptides (CPPs) are widely used carrier molecules for intracellular protein delivery. While CPPs can be assembled with protein cargos via different methods including genetic fusion, chemical ligation and physical complexation, each method has its own limitations. Inspired by our previous findings that structurally constrained small synthetic CPPs are highly efficient delivery carriers, we here developed a new protein delivery approach through which structurally constrained CPPs were genetically encoded into a protein of interest, thus to achieve high delivery efficiency, CPP stability and ease for production at the same time. The primary goal of my dissertation research is to engineer proteins of interest with intrinsic cell-permeability, by applying the technique that structurally constrained CPPs were incorporated into protein loop regions, resulting in functional delivery of different protein cargos inside the cells. We first identified several active CPP sequences by designing and characterizing a small group of cyclic peptide library. The insertion of selected CPPs into a solvent exposed loop of enhanced green fluorescence protein (EGFP) resulted in efficient intracellular delivery. We then applied this strategy to intracellular delivery of purine nucleoside phosphorylase (PNP), a protein missing in immunodeficiency diseases. Efficient delivery of PNP by loop-inserted CPP restored cytosolic enzyme activity in PNP deficient cells and corrected their metabolic defect, indicating significant therapeutic values of this approach. Next, to further enhance protein delivery efficiency, we incorporated non-proteinogenic building blocks into the loop-inserted CPPs via cysteine derivatization. Through screening a small library of different hydrophobic moieties, the cell-penetrating activity of engineered EGFP was increased by up to 15-fold. Finally, we successfully delivered an antibody fragment (nanobody) into the cells by inserting CPP sequence into one of its CDR loops. The engineered GFP binding nanobody (GBN) retained high antigen binding affinity and was able to selectively engage its antigen after cytosolic entry. These findings established a general approach to produce cell-permeable antibody fragments for intracellular targets.