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Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule

Authors
  • Behzadipour, Yasaman
  • Hemmati, Shiva1, 2
  • 1 Biotechnology Research Center, Shiraz University of Medical Sciences, Shiraz 71345-1583, Iran
  • 2 Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz 71345-1583, Iran
Type
Published Article
Journal
Molecules
Publisher
MDPI AG
Publication Date
Nov 26, 2019
Volume
24
Issue
23
Identifiers
DOI: 10.3390/molecules24234318
PMID: 31779220
PMCID: PMC6930620
Source
PubMed Central
Keywords
License
Green

Abstract

Access of proteins to their intracellular targets is limited by a hydrophobic barrier called the cellular membrane. Conjugation with cell-penetrating peptides (CPPs) has been shown to improve protein transduction into the cells. This conjugation can be either covalent or non-covalent, each with its unique pros and cons. The CPP-protein covalent conjugation may result in undesirable structural and functional alterations in the target protein. Therefore, we propose a systematic approach to evaluate different CPPs for covalent conjugations. This guide is presented using the carboxypeptidase G2 (CPG2) enzyme as the target protein. Seventy CPPs —out of 1155— with the highest probability of uptake efficiency were selected. These peptides were then conjugated to the N - or C-terminus of CPG2. Translational efficacy of the conjugates, robustness and thermodynamic properties of the chimera, aggregation possibility, folding rate, backbone flexibility, and aspects of in vivo administration such as protease susceptibility were predicted. The effect of the position of conjugation was evaluated using unpaired t-test ( p < 0.05). It was concluded that N-terminal conjugation resulted in higher quality constructs. Seventeen CPP-CPG2/CPG2-CPP constructs were identified as the most promising. Based on this study, the bioinformatics workflow that is presented may be universally applied to any CPP-protein conjugate design.

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