Expression of Cell-Penetrating Peptides Fused to Protein Cargo

Abstract

Cell-penetrating peptides (CPPs) are short peptides that can cross cell membranes. CPPs enable the delivery of biomolecules into cells and can act as drug-delivery vectors. Because recombinant production of CPPs as fusions to protein “cargo” leads to low yields for some CPP-cargo fusions, approaches to enhance the recombinant expression of peptide-cargo fusions need to be identified. We optimized expression conditions in Escherichia coli for fusions of CPPs (SynB, histatin-5, and MPG) to the cargo proteins biotin carboxyl carrier protein, maltose-binding protein, and green fluorescent protein. We used Western blotting to evaluate induction temperatures of 37, 30, and 20°C, and induction times of 6, 10, and 24 h. Glutathione-S-transferase was incorporated as a fusion partner to improve expression. In general, expression at 37°C for 6 and 10 h led to the highest levels of expression for the different CPP-cargo constructs. The improvements in expression of CPP-cargo fusions will allow higher yields of CPP-cargo fusions for studies of their translocation into cells.

Advances in the use of plants as potential biofactories in the production of antimicrobial peptides

“…In bacterial expression systems, the fusion can be necessary for bacterial protection. [31,[55][56][57][58][59] However, these processes require the subsequent removal of the fusion partner, which can constitute a high cost in production process. On the other hand, plants can represent attractive biofactories to produce fused recombinant AMPs, considering that plants cannot be an AMP target.…”

Asymmetric Assembly and Self-Adjuvanted Antigen Delivery Platform for Improved Antigen Uptake and Antitumor Effect

“…To facilitate the subsequent display of other peptides or proteins of interest, we employed a Plug-and-Display strategy based on the SpyCatcher/SpyTag system , to enable rapid preparation of vaccines. In addition, the use of a Plug-and-Display strategy allows for the avoidance of reduced protein yields that may result from direct fusion expression . Considering that the CPPs are short and randomly arranged on the surface of BLS with other large-size antigens may affect their membrane penetration efficiency due to spatial resistance.…”

“…In addition, the use of a Plug-and-Display strategy allows for the avoidance of reduced protein yields that may result from direct fusion expression. 13 Considering that the CPPs are short and randomly arranged on the surface of BLS with other large-size antigens may affect their membrane penetration efficiency due to spatial resistance. Therefore, we used the BLS asymmetric assembly design 14 to arrange OVA peptides and KALA on both sides of the BLS to achieve complete exposure of CPPs.…”

Design of Peptide-Based Protein Degraders via Contrastive Deep Learning

“…Peptides have been widely recognized as a more selective, effective, and safe method for targeting pathogenic proteins, due to their sequence-specific binding to regions of partner molecules [Padhi et al, 2014, Buchwald et al, 2014]. They have further demonstrated targeting of both extracellular and intracellular proteins, due to their small size and enhanced permeability, with or without conjugation to cell penetrating peptide (CPP) sequences [Lindgren et al, 2000, Lozano et al, 2017, Adhikari et al, 2018]. Beyond standalone peptide binders, our group has recently fused computationally-designed peptides to effector domains, such as E3 ubiquitin ligases, to enable binding and selective intracellular degradation of pathogenic targets of interest [Chatterjee et al, 2020].…”