Abstract
Sense and antisense oligonucleotide pairs encoding cell-penetrating peptides PTD (Tat47–57), DPV3A, E162, pVEC, R11, and TP13 were used to construct two sets of pET22b-CPP-DsRed and pET22b-CPP-J-DsRed vectors for CPP-DsRed and CPP-J-DsRed recombinant proteins expression. PTD-DsRed, DPV3A-DsRed, PTD-J-DsRed, and DPV3A-J-DsRed recombinant proteins were expressed in a soluble form. PTD-J-DsRed and DPV3A-J-DsRed recombinant proteins were able to escape from E. coli host cells into the culture medium. The membrane-penetrating activity of PTD-J-DsRed and DPV3A-J-DsRed recombinant proteins to mammalian cells was more effective than that of PTD-DsRed and DPV3A-DsRed. The route of the cellular membrane translocation of these recombinant proteins is suggested via macropinocytosis followed by an endosomal escape pathway.
Highlights
The primary amphipathic Cell-Penetrating Peptides (CPPs) such as transportan [11] and TP10 [12] are usually longer than 20 amino acids with periodically hydrophobic and hydrophilic residues along the primary sequence
After transformation with pET22b-CPP-DsRed plasmids, E. coli Rosetta gamiB(DE3)pLysS cells were cultured in 2x YT medium supplemented with 0.4% glucose and antibiotics until OD600 reached 0.6
PTD- and DPV3A-DsRed recombinant proteins were found in the soluble fraction; on the other hand, TP13, E162, and pVEC-DsRed recombinant proteins were found in the insoluble fraction
Summary
A 16-amino acid peptide derived from the third helix of the homeodomain of Antennapedia, termed as penetratin, was found to translocate through cell membrane as well [7]. CPPs, either protein-derived or chemically synthesized, can be categorized into primary amphipathic, secondary amphipathic, and nonamphipathic [10]. The primary amphipathic CPPs such as transportan [11] and TP10 [12] are usually longer than 20 amino acids with periodically hydrophobic and hydrophilic residues along the primary sequence. In comparison to the primary amphipathic CPPs, the secondary amphipathic CPPs such as penetratin, pVEC [13], and E162 [14] contain less amino acid residues and perform amphipathic structure upon interacting with phospholipid membrane. The third class CPPs, such as R8 [15], DPV3 [16], and PTD [4], are relatively short and contain very high content of arginine
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