Translocation of Cell Penetrating Peptides on Chlamydomonas reinhardtii Arumuganainar Suresh, Yeu-Chun Kim Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, Republic of Korea; telephone: þ82-42-350-3939; fax: 42-350-3910; e-mail: dohnanyi@kaist.ac.kr ABSTRACT: Engineering of algal cells by delivering macro- molecules through cell wall and plasma membrane presents many difficulties with the conventional methods. Recent research has shown that a new delivery method, namely cell penetrating peptide (CPP), has the ability to translocate into animal, plant, fungal, and bacterial cells. This study reports the apparent translocation of CPPs into algal cells of Chlamydomonas reinhardtii and the successful delivery of the conjugated fluorochrome. Although translocation efficiency was specific to each CPP studied, pVEC (peptide vascular endothelial cadherin) showed the highest translocation efficiency in comparison with penetratin (PEN), trans- activating transcriptional (TAT) peptide, and transportan (TRA). The maximum translocation of pVEC into the algal cell was reached in 15 min of incubation at 25  C. More importantly, translocation with pVEC demonstrated an absence of cytotoxicity. Thus, we suggested that pVEC is an attractive candidate for delivering macromolecules into algal cells for use in industrial applications. Biotechnol. Bioeng. 2013;110: 2795–2801. ß 2013 Wiley Periodicals, Inc. KEYWORDS: cell penetrating peptides; delivery vector; microalgae; pVEC; translocation Inducing the uptake of macromolecules by cells has been widely perceived as a difficult process, because the existence of plasma membranes (hydrophobic) and cell walls normally prevents the passage of macromolecules (hydrophilic) into cells, unless specific mechanisms are employed to bypass these barriers, such as receptor, transporter, or endocytosis. Protein transduction domains (PTDs), also called cell penetrating peptides (CPPs) are small (<30 amino acids) peptides which are rich in basic amino acids and were originally identified from the trans-activating transcriptional (TAT) protein of human immunodeficiency virus (HIV-1) (Frankel and Pabo, 1988; Green and Loewenstein, 1988) and Drosophila Antennapedia homeoprotein (penetratin) (Joliot et al., 1991). These cationic peptides can penetrate cell membrane in an energy and endocytosis independent fashion (Vives et al., 1997). In addition, they can carry wide varieties of biologically active macromolecules into mammalian cells with higher efficiency, specific targeting, and most impor- tantly absence of cytotoxicity (Fenton et al., 1998; Snyder and Dowdy, 2005). Currently, many synthetic CPPs are employed to deliver different macromolecules such as peptides, proteins, nucleic acids, liposomes, carbohydrates, drugs, nanoparticles, fluorochrome, and quantum dot into mam- malian cells (Joliot and Prochiantz, 2004; Schwarze et al., 1999; Snyder and Dowdy, 2005) for therapeutic and other clinical applications. While most of the translocation studies up-to-date have been done in mammalian cells, very few studies have been executed in non-mammalian cells, such as plants (Chugh and Eudes, 2008), fungi (Holm et al., 2005), bacteria (Liu et al., 2008), and microalgae (Hyman et al., 2012). Lately, the scientific community realized that CPP is a powerful tool for delivering macromolecules across cell walls (bacteria, algae, plant) and cell membranes in diverse cells for basic and advanced research. In this study, the author splaced special importance on investigating the potential of CPPs in algal cells of C. reinhardtii. Although many techniques such as glass beads, electroporation, gene gun and viral vectors are available for use as delivery methods in algae (Henry and Meints, 1994), these methods are relatively inefficient, tedious, and time-consuming due to the lack of the ability to overcome the cell wall and various extracellular polymers. It is clear that there is an urgent need for the emergence of a better transformation delivery tool for enhancing biofuel production from microalgae. Thus, the aim of this study was directed towards the possibility of using CPP as a delivery tool in microalgae. We observed very clear translocation of four selected FITC conjugated CPPs (Table I) into algal cells of C. reinhardtii (Figs. 1–4) and free FITC is not, however the uptake efficiency Correspondence to: Yeu-Chun Kim Contract grant sponsor: Advanced Biomass R&D Center (ABC), Korea Contract grant sponsor: Ministry of Education, Science and Technology, Korea Contract grant number: N01120759 Received 12 March 2013; Accepted 5 April 2013 Accepted manuscript online 24 April 2013; Article first published online 30 April 2013 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.24935/abstract). DOI 10.1002/bit.24935 COMMUNICATION TO THE EDITOR ß 2013 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 110, No. 10, October, 2013 2795