Cell penetrating peptides (CPPs), also referred to as protein transduction domains (PTDs), are polypeptide domains that have the ability to penetrate biological cell membrane. Previously, CPPs have been studied to deliver macromolecular cargos such as peptides, proteins, antisense oligonucleotides, ...
Cell penetrating peptides (CPPs), also referred to as protein transduction domains (PTDs), are polypeptide domains that have the ability to penetrate biological cell membrane. Previously, CPPs have been studied to deliver macromolecular cargos such as peptides, proteins, antisense oligonucleotides, and small interfering RNAs, etc. TAT, VP22, Antp, and R9 have been widely studied for CPP based therapeutic applications. However, these CPPs are non-human originated or synthetic peptides, with disadvantage of potential toxicity and immunogenicity. To overcome this hurdle, here we identified a novel CPP (AP) from human neuronal protein, Astrotactin1 (ASTN1). AP-EGFP recombinant DNAs were constructed and the proteins were expressed in bacterial system and purified by Ni-NTA affinity chromatography and PD-10 desalting column. We determined the transduction efficiency of purified AP-EGFP proteins in human leukemia Jurkat T cells. AP-EGFP showed dramatic intracellular transduction efficiency significantly more than TAT and R9. Next, we attempted to optimize the AP-CPP sequence by deletion of amino acid or tandem repeat sequence. From the results, AP-sequence was concluded as optimal length. In order to investigate critical amino acid, which contributes AP’s dramatic delivery efficiency, we tested various mutant forms. We found that cysteine is essential to AP’s intracellular transduction efficiency. Interestingly, addition of one cysteine residue also improved transduction efficiency of NP2. Cystein residue allows the CPP to dimerize by disulfide bond formation, possibly increasing its affinity to cell membrane. Its transduction mechanism was investigated that AP-EGFP could enter into the cells by energy dependent mechanism such as endocytosis. Also, AP-EGFP could penetrate cell membrane after binding to extracellular heparan sulfate proteoglycans by charge interaction. Next, intracellular localization of AP-EGFP was investigated that it was localized into the nucleus as well as cytoplasm in HeLa cells after two hours incubation suggests AP-CPP can be used for delivery of nuclear or cytoplasmic cargos for the therapeutic purposes. Finally, we attempted to deliver EGFP protein in vivo that AP-EGFP has successfully delivered into the mouse organs including spleen, intestine, liver, lung, heart, and even brain after intra-peritoneal injection. AP-EGFP was rapidly penetrated into the organs within 30 minutes and the maximum fluorescent was detected until 2 hours. The fluorescent was disappeared after 6 hours implies the half-life of delivered protein. Therefore, this study demonstrates that a novel human derived AP-CPP sequence would have an advantage over the limitation of non-human originated CPPs. And its potent efficiency was derived from dimerization by cysteine residue suggesting addition of cysteine to any CPP sequence would increase its delivery efficiency without possible side effects such as toxicity. We expect that AP-CPP can be used for the delivery of intracellular therapeutic bio-molecules in various human diseases.
Cell penetrating peptides (CPPs), also referred to as protein transduction domains (PTDs), are polypeptide domains that have the ability to penetrate biological cell membrane. Previously, CPPs have been studied to deliver macromolecular cargos such as peptides, proteins, antisense oligonucleotides, and small interfering RNAs, etc. TAT, VP22, Antp, and R9 have been widely studied for CPP based therapeutic applications. However, these CPPs are non-human originated or synthetic peptides, with disadvantage of potential toxicity and immunogenicity. To overcome this hurdle, here we identified a novel CPP (AP) from human neuronal protein, Astrotactin1 (ASTN1). AP-EGFP recombinant DNAs were constructed and the proteins were expressed in bacterial system and purified by Ni-NTA affinity chromatography and PD-10 desalting column. We determined the transduction efficiency of purified AP-EGFP proteins in human leukemia Jurkat T cells. AP-EGFP showed dramatic intracellular transduction efficiency significantly more than TAT and R9. Next, we attempted to optimize the AP-CPP sequence by deletion of amino acid or tandem repeat sequence. From the results, AP-sequence was concluded as optimal length. In order to investigate critical amino acid, which contributes AP’s dramatic delivery efficiency, we tested various mutant forms. We found that cysteine is essential to AP’s intracellular transduction efficiency. Interestingly, addition of one cysteine residue also improved transduction efficiency of NP2. Cystein residue allows the CPP to dimerize by disulfide bond formation, possibly increasing its affinity to cell membrane. Its transduction mechanism was investigated that AP-EGFP could enter into the cells by energy dependent mechanism such as endocytosis. Also, AP-EGFP could penetrate cell membrane after binding to extracellular heparan sulfate proteoglycans by charge interaction. Next, intracellular localization of AP-EGFP was investigated that it was localized into the nucleus as well as cytoplasm in HeLa cells after two hours incubation suggests AP-CPP can be used for delivery of nuclear or cytoplasmic cargos for the therapeutic purposes. Finally, we attempted to deliver EGFP protein in vivo that AP-EGFP has successfully delivered into the mouse organs including spleen, intestine, liver, lung, heart, and even brain after intra-peritoneal injection. AP-EGFP was rapidly penetrated into the organs within 30 minutes and the maximum fluorescent was detected until 2 hours. The fluorescent was disappeared after 6 hours implies the half-life of delivered protein. Therefore, this study demonstrates that a novel human derived AP-CPP sequence would have an advantage over the limitation of non-human originated CPPs. And its potent efficiency was derived from dimerization by cysteine residue suggesting addition of cysteine to any CPP sequence would increase its delivery efficiency without possible side effects such as toxicity. We expect that AP-CPP can be used for the delivery of intracellular therapeutic bio-molecules in various human diseases.
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