A peptide carrier for the delivery of biologically active proteins into mammalian cells

Abstract

The development of peptide drugs and therapeutic proteins is limited by the poor permeability and the selectivity of the cell membrane. There is a growing effort to circumvent these problems by designing strategies to deliver full-length proteins into a large number of cells. A series of small protein domains, termed protein transduction domains (PTDs), have been shown to cross biological membranes efficiently and independently of transporters or specific receptors, and to promote the delivery of peptides and proteins into cells. TAT protein from human immunodeficiency virus (HIV-1) is able to deliver biologically active proteins in vivo and has been shown to be of considerable interest for protein therapeutics. Similarly, the third alpha-helix of Antennapedia homeodomain, and VP22 protein from herpes simplex virus promote the delivery of covalently linked peptides or proteins into cells. However, these PTD vectors display a certain number of limitations in that they all require crosslinking to the target peptide or protein. Moreover, protein transduction using PTD-TAT fusion protein systems may require denaturation of the protein before delivery to increase the accessibility of the TAT-PTD domain. This requirement introduces an additional delay between the time of delivery and intracellular activation of the protein. In this report, we propose a new strategy for protein delivery based on a short amphipathic peptide carrier, Pep-1. This peptide carrier is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. In addition, this peptide carrier presents several advantages for protein therapy, including stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum. Pep-1 technology should be extremely useful for targeting specific protein-protein interactions in living cells and for screening novel therapeutic proteins.

Real‐Time Cellular Imaging of Protein Poly(ADP‐ribos)ylation

“…The uptake of the negatively charged NAD + across the cell membrane was achieved by using the carrier peptide Pep 1. [16] We first tested different cargo/carrier ratios and analyzed the labeling efficiency by flow cytometry. In addition, cell viability was estimated by Sytox Blue staining under varying conditions.…”

Microenvironmental regulation of biomacromolecular therapies

“…Various recombinant growth factors and antibodies are being used to elicit specific cellular activities that are useful for wound healing and regeneration of tissues and organs, or to limit the pathogenesis and metastasis of malignant cells (reviewed elsewhere) [1][2][3][4][5] . Different growth factors may also be combined to attain synergistic improvements in therapeutic efficiency 52,53 .…”

“…A broad array of therapeutic biomacromolecules including proteins [1][2][3][4][5] , plasmid DNAs (pDNAs) 6 and various forms of RNA have been identified 7,8 , engineered and used in various clinical trials and approved products 9,10 . The completion of the Human Genome Project will probably accelerate the discovery and application of biomacromolecular therapies.…”

“…For example, therapeutic proteins and nucleotides have been hybridized through chemical fusion or complexation with diverse targeting 17,18 and delivery molecules to enhance infiltration into tissues and cells 19,20 and tissuespecific localization [21][22][23][24] . Furthermore, therapeutic biomacromolecules have been loaded into various biomaterials to enable a sustained and localized delivery manner while preserving bioactivity, as extensively reviewed elsewhere 5,[25][26][27][28][29][30] .…”

Commentary on “Copper chaperone for Cu,Zn-SOD supplement potentiates the Cu,Zn-SOD function of neuroprotective effects against ischemic neuronal damage in the gerbil hippocampus”

“…In this study, Hwang and colleagues used the Pep-1 peptide transduction domain [25] to show that ischemic neuronal damage could be attenuated by Pep-1-CuZn-SOD and that the protection was enhanced if CCS was given in addition to CuZn-SOD. These results were significant because they demonstrated that CuZn-SOD has a stronger neuroprotective effect when it is co-administered with its chaperone.…”