Identification of Novel Cell-Penetrating Peptide Derived from Human Protein NLBP as Drug Delivery System

Abstract

Cell penetrating peptides (CPPs) have the ability to penetrate cellular membranes and various CPPs have been identified for the delivery of biomolecules. In previous studies, although various CPPs such as TAT, VP22 and antennapedia (Antp) are proved as the high efficacy CPP for cell infiltration, these non-human originated CPPs have the limitations of potential toxicity and immunogenicity. To tackle this problem, here first we have newly identified novel CPPs (NP1, NP2) from amino acid sequences of human proteins NLBP (Novel LZAP binding protein). NP-EGFP DNAs were cloned into a protein expression vector and purified proteins were tested in Jurkat and HeLa cells to prove transduction efficiency. All NP-EGFPs were delivered into the cells in a dose and time manner and localized into the nucleus as well as cytoplasm. In addition, we further optimized the NP-CPP sequence by design the tandem repeat of each sequence (dNP1, dNP2). Especially dNP2 sequence showed much increased transduction ability than any other CPPs including TAT, R9 or Hph-1. These NLBP derived CPPs penetrate cell membrane through binding to heparan sulfate by charge interaction and lipid raft mediate endocytosis. Interestingly, dNP2-EGFP was significantly delivered into primary human cell and mouse cells while other CPPs had only marginal transduction efficiency. Also, it could be delivered into various tissues including spleen, intestine, liver, lung, heart, and even brain in mice after intraperitoneal injection. Therefore, this study suggests that dNP2 sequence is finally optimized from human protein NLBP and this novel human derived CPP would have advantage as drug delivery system, over the limitation of non-human originated CPPs. Furthermore, we expect that these CPPs can be used for the therapeutic application through further studies.