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A plasmid display platform for the selection of peptides exhibiting a functional cell-penetrating phenotype

A plasmid display platform for the selection of peptides exhibiting a functional cell-penetrating phenotype
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  A Plasmid Display Platform for the Selection of Peptides Exhibiting a FunctionalCell-Penetrating Phenotype Shan Gao Dept. of Chemical Engineering, Columbia University in the City of New York, New York, NY 10027 Melissa J. Simon and Barclay Morrison III Dept. of Biomedical Engineering, Columbia University in the City of New York, New York, NY 10027 Scott Banta Dept. of Chemical Engineering, Columbia University in the City of New York, New York, NY 10027  DOI 10.1002/btpr.490 Published online October 11, 2010 in Wiley Online Library ( Cell-penetrating peptides (CPPs) represent a promising nonviral platform for the delivery of therapeutic cargos to cells and tissues. However, these peptides are often nonspecific, and their mechanism of action is still a subject of debate, which hinders the design of new CPPs. The al-ternative to rational protein design is the combinatorial approach to protein engineering,whereby large libraries of peptides are created and a screening or selection procedure is used to identify members with the desired phenotype(s). Here we describe a novel procedure for selecting peptides with a CPP phenotype using a plasmid display (PD) platform to link the pep-tides to their encoding DNA sequences. The PD system is based on genetic fusions to a DNAbinding domain. The plasmid was designed to concomitantly express a fluorescent reporter  protein to serve as a mock therapeutic cargo indicating its functional delivery into a cell. Wehave demonstrated this selection strategy using a control CPP (the TAT peptide) in the PC12neuronal-like cell line. In the absence of transfection reagents, TAT was unable to deliver the protein/DNA complexes. The inclusion of the HA2 peptide from the hemagglutinin protein and the addition of polyethylenimine (PEI) were similarly ineffective. The addition of Lipofect-amine, however, enabled the TAT-mediated delivery of the protein/DNA complexes, which wassignificantly better than control experiments without a CPP. This new PD selection platformwill be a valuable new approach for use in identifying unique CPPs from randomized librarieswith novel abilities and specificities.  V V C  2010 American Institute of Chemical Engineers  Bio-technol. Prog.,  26: 1796–1800, 2010  Keywords: cell-penetrating peptides, plasmid display, functional selection, TAT  Introduction There is a critical need for new methods to deliver thera-peutics across the plasma membrane, and cell-penetratingpeptides (CPPs) are a potential solution to this challenge. 1–6 These peptides are usually short, mostly basic sequenceswhich can be readily conjugated to a variety of cargos,including proteins, DNA, RNA, small molecules, and nano-particles. 7–11 Although the mechanism of penetration is stillbeing elucidated, 3,5,12 many research groups are working todevelop CPPs for use in therapeutic applications. The use of CPPs in clinical applications, however, has been hamperedby several limitations, including inefficient delivery and lackof specificity of these molecules for different cell- and tis-sue-types. 13–16 Combinatorial techniques in protein engineering, such asdirected evolution, represent a valuable approach for theidentification of new efficient and specific CPPs. The mainchallenge for the application of these methods is the diffi-culty in identifying and separating the peptides that enter thecytoplasm and perform desired cellular functions from thosethat remain outside the cell, remain trapped in endosomes, or are otherwise rendered ineffective. There is a substantialneed to develop new selection procedures that can clearlyidentify CPPs from a library of peptides that have the abilityto cross the plasma membrane and functionally deliver acargo to the cytoplasm.Combinatorial protein engineering methods require a link-age between the library peptides and their associated DNAsequences for subsequent identification after screening or selection. 17,18 Phage display is frequently used for this pur-pose, but there are alternative methods. Plasmid display (PD)is a conceptually simple display method that uses a noncova-lent linkage between the DNA sequence and the displayedprotein via a modified DNA binding protein (Figure 1). Sev-eral PD constructs have been reported based on differentDNA binding proteins, including the  lac  repressor, 19 theGAL4 DNA binding domain, 20 and the NF- j B p50 DNAbinding domain. 6,21 One of the advantages of a PD system is Correspondence concerning this article should be addressed to S.Banta at 1796  V V C 2010 American Institute of Chemical Engineers  that the plasmid itself can be bifunctional in that it carriesthe DNA sequence of the candidate CPP while also servingas the cargo that will be delivered. An appropriate gene canbe inserted into the plasmid, and its expression unequivo-cally demonstrates intracellular delivery of the cargo in afunctional form.In this study, we report the development of a PD systembased on the p50 DNA-binding domain that can be used for selection of functional CPPs. The well-known TAT CPP wasused as a positive control, and a fluorescent transgene[enhanced yellow fluorescent protein (EYFP)] was added tothe plasmid to serve as a model cargo. The ability of theTAT peptide to deliver the PD complex to neuronal-likePC12 cells was investigated. It was discovered that a charge-shielding reagent was required for the CPP to deliver theprotein/DNA complexes. These results suggest that the PDsystem will be a valuable platform for the identification of novel CPPs in future combinatorial selection projects. Materials and Methods The pRES115 vector containing the p50 gene and the tar-get- j B sequence (GGGAATTCCC) was kindly provided byDr. Jonathan Blackburn at University of the Western Cape,South Africa. 21 Four plasmid vectors, pTAT, pCON, pHA2-TAT, and pHA2-CON (Figure 1), were constructed by clon-ing necessary sequences into the pRES115 vector. 22 Theamino acid sequence of the TAT peptide wasGYGRKKRRQRRRG, and the amino acid sequence of theHA2 peptide was GLFEAIEGFIENGWEGMIDGWYG.Protein/DNA complexes made from the four plasmidswere expressed with minor modification of the method previ-ously described. 21 The protein/DNA constructs were purifiedusing Ni-NTA immobilized metal affinity chromatographyresin (Qiagen) at 4  C. The column elutions were concen-trated using Amicon YM-10 columns (Millipore). The finalconcentrated protein/DNA samples were frozen and used for transfection within a week. The purified protein/DNA con-structs were named pdTAT, pdCON, pdHA2-TAT, andpdHA2-CON. The concentration of the plasmid in each of the samples was measured using quantitative PolymeraseChain Reaction (q-PCR) (Bio-Rad Laboratories). StandardSodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis(SDS-PAGE), Western blotting, native gel electrophoresis,and agarose gel electrophoresis experiments were performedto monitor the presence and purity of the protein/DNA com-plexes during the purification process.PC12 cells (The American Type Culture Collection,ATCC) were cultured as previously described. 4 The cellswere grown for 1 day before being used for transfectionexperiments. PC12 cells in a single well from a 24-well platewere transfected using varying amounts of the protein/DNAcomplexes, pdTAT, pdCON, pdHA2-TAT, and pdHA2-CON. Lipofectamine-assisted transfection of pdTAT,pdCON, or neat pCON plasmid (as a control) was performedusing 10  l L of Lipofectamine 2000 (Invitrogen) followingthe manufacturer’s protocol. For PEI-assisted transfection, abranched polyethylenimine (PEI,  M  w  25,000 Da, Sigma-Aldrich) was used. Varying amounts of the PEI solutionwere incubated with pCON neat plasmid, pdTAT or pdCONcomplexes at varying  N   /   P  ratios before incubation with cells.After incubating the sample with PC12 cells for 4 h, thetransfection medium was exchanged with fresh growth me-dium and the cells were cultured for an additional 20–24 hto enable the expression of EYFP.The cells from the transfection experiments were trypsi-nized, pelleted, and resuspended in Phosphate Buffered Sa-line (PBS) buffer for analysis using a FACSAria cell sorter or FACSCanto II flow cytometer (Becton Dickinson, Frank-lin Lakes, NJ). The cells with expressed yellow fluorescentprotein were detected through the FITC channel (ex 488, em515–545). The gates were set using untreated PC12 cells andthe cells transfected with neat plasmid using Lipofectamine2000. The delivery efficiency was evaluated as the percent-age of viable, fluorescent cells.Statistical differences between the delivery of pdTAT andpdCON using Lipofectamine were calculated using Student’s t  -test with  P \ 0.05 being considered significant. Results and Discussion Once the new PD constructs were created and verified, theprotein/DNA complexes were expressed and purified. Thepurification of the complexes was challenging, as the yieldof the complexes was very low, by design. The developmentof the PD system required the  Lac  promoter in the pRES115vector to be modified by inserting a target- j B bindingsequence into the promoter. 21,23 This was done in an attemptto reduce the expression of the DNA-binding protein oncethe p50 homodimer bound the plasmid. Ideally, this wouldlead to a close ratio of homodimeric protein to DNA (Figure1), thus eliminating large amounts of uncomplexed protein.We verified the coexistence of the fusion protein and theplasmid in the purified samples using a battery of protein(SDS-PAGE, native gels, and Western blots) and DNA(PAGE, agarose gels) electrophoresis experiments (data notshown). However, the yield of the purified complexes, basedon the amount of plasmid of the complexes calculated fromq-PCR results, was about 30–40 ng from 1 L culture eventhough the expression and purification protocol had beenmodified to increase the yield about sixfold to eightfold. Inaddition, there appeared to be significant protein and DNAcontamination in the purified samples. Previously, it hasbeen shown that selections with the PD system could be per-formed in the  Escherichia coli  cytoplasmic milieu withoutpurification. 21,24 It was not clear if the contaminating pro-karyotic material would adversely affect the transfection of mammalian cells.The partially purified pdTAT and pdCON protein/DNAcomplexes were incubated with PC12 cells at varying con-centrations, but the expression of the EYFP gene was notobserved using either microscopy or flow cytometry. Cellular toxicity was not observed, which indicated that the level of purification of the protein/DNA complexes was sufficient for these transfection experiments. Control experiments with thepurified plasmids (pCON and pTAT) alone combined withthe Lipofectamine transfection reagent did result in fluores-cent cells, which indicated that the EYFP genes in the plas-mids were functional. In an attempt to enhance the deliveryof the pdTAT and pdCON protein/DNA complexes, the HA2peptide derived from influenza virus hemagglutinin proteinwas genetically appended to the N-terminus of the p50 pro-teins because it has been shown that the HA2 peptide facili-tates the disruption of endosomes, thereby increasing theefficiency of TAT-mediated delivery of protein and siRNAcargos. 25–27 The HA2 peptide was inserted just after the His Biotechnol. Prog.,  2010, Vol. 26, No. 6  1797  and FLAG tags, immediately upstream of the p50 gene.However, detectable fluorescent cells were again notobserved after incubation with purified pdHA2-TAT andpdHA2-CON complexes. Because the addition of the HA2was unable to facilitate delivery, the limitation of the systemis likely the inability of the TAT peptide to deliver the largeprotein/DNA complexes across the plasma membrane andinto the cells and not endosomal entrapment.The observation that the two copies of the TAT peptidewere unable to deliver the protein/DNA complexes to thePC12 cells was not totally unexpected. In previously pub-lished work using TAT-based peptides or polymers for plas-mid delivery, the molar ratio of peptide to plasmid rangedfrom hundreds to thousands for detectable delivery. 28–32 Inaddition, Kilk et al. attempted to deliver plasmid DNA usingthe TP10 CPP tethered with a peptide nucleic acid linker atthe molar ratio of peptide to DNA of 1:1. 33 By itself, thisconstruct also failed to deliver the plasmid DNA to cells.The addition of polyethylenimine (PEI), however, was ableto facilitate the delivery of the plasmid, but the efficiencywas very low. In previous work reported by our group, 6 asignificant amount of a TAT-containing fusion protein p50-GFP-TAT (molar ratio of protein to plasmid at 8400:1) wasrequired to deliver a plasmid containing a red fluorescenttransgene without the aid of a transfection reagent. Wehypothesized that the CPP-mediated delivery of the protein/ DNA PD complexes failed due to the pronounced negativecharges on the plasmid which interact with positivelycharged TAT sequence, thereby preventing proper interactionof the CPPs with the cell membrane.To address this problem, we explored charge-shieldingreagents to neutralize some of the negative charges on theplasmid. We first chose PEI, which is a polycationic polymer commonly used for DNA transfection. Branched PEI with a  M  w  of    25 kDa has been shown to be efficient for plasmiddelivery. 34 We studied the correlation of the  N   /   P  ratio (molar ratio of the amine groups with positive charges of PEI to thephosphate groups with negative charges of DNA) and thedelivery efficiency of the neat pCON plasmid (data notshown). An  N   /   P  ratio of 12:1 was found to be optimal for the delivery of the neat pCON plasmid to the PC12 cells.PEI was added to the purified protein/DNA complexes at aratio of 12:1 and a ratio of 6:1 based on the amount of plas-mid present in the samples as obtained by q-PCR. The addi-tion of PEI did not enhance the delivery of the pdTAT or pdCON complexes as compared to the untreated cells (Fig-ure 2a). A likely explanation for this is the presence of sig-nificant amounts of protein and DNA in the partially purifiedprotein/DNA samples. The efficiency of PEI is quite sensi-tive to the  N   /   P  ratio, and additional proteins and DNA mole-cules in the sample would significantly alter the  N   /   P  ratiofrom the calculated values based on the plasmid concentra-tion measured by q-PCR.Because PEI at the optimal ratio failed to enhance thedelivery of the pdTAT and pdCON complexes, we testedLipofectamine as a charge neutralization agent for the plas-mid in the complexes (Figure 2b). The addition of the Lipo-fectamine reagent enabled the cell penetrating ability of theTAT peptides, as there was a statistically significant, Figure 2. Polyethylenimine (PEI) (a) or Lipofectamine (b)mediated delivery of pdCON and pdTAT protein/DNA complexes to PC12 cells. pdCON or pdTAT samples containing 50 ng of plasmid wereincubated with branched PEI (  M  w  25,000 Da,  N   /   P  ¼  6 or 12)or 10  l L Lipofectamine before addition to the cells. Cells wereanalyzed by flow cytometry 20–24 h after transfection. Theresults are expressed as percentage of cells with yellow fluores-cence as detected through a FITC channel ( n    3, error barsrepresent SE). The gate was set based on untreated cells andthe cells transfected with the neat pCON plasmid using theLipofectamine reagent. The blank samples in both figures rep-resent untreated cells. The ‘‘*’’ indicates the value with pdTATis significantly different from the cells treated with pdCONusing Student’s  t  -test (  P \ 0.05). Figure 1. Schematic diagrams of the pTAT plasmid and thepdTAT protein/DNA complex. pTAT (Left) was created from the pRES115 PD vector byintroducing the TAT sequence to the C-terminus of the p50gene, His and FLAG tags to the N-terminus of the p50 gene,and the complete enhanced yellow fluorescent protein (EYFP)gene into the plasmid. pdTAT (Right) is the protein/DNA com-plex that was generated when the expressed fusion protein(named HPT) forms a homodimer that binds to a specific DNAsequence (target  j B) located in the promoter region for the p50fusion protein. The pCON plasmid was created by adding astop codon at the end of the p50 protein, and the HA2 contain-ing plasmids (pHA2-TAT and pHA2-CON) were created byinserting the HA2 sequence between the His and FLAG tagsand the p50 gene in the pTAT and pCON vectors, respectively. 1798  Biotechnol. Prog.,  2010, Vol. 26, No. 6  approximately threefold difference in the transfection effi-ciencies between pdTAT and pdCON. Interestingly, thetransfection efficiencies with pdTAT and pdCON were far lower than what was observed under identical conditionswith the neat pCON plasmid (50.8%    3.5%), which indi-cates that the addition of the p50 protein (and/or the contam-inating proteins and DNA) inhibit Lipofectamine-mediatedtransfection. However, because all selection experimentswould be performed in the same background of cationicpolymer, contaminating protein and DNA, it is the signifi-cant difference in delivery efficiency engendered by theinclusion of the CPP peptide that will be of interest.Our encouraging results indicate that it should be possibleto isolate peptides with cell penetrating activity away fromthose without a CPP phenotype. This selection should allowCPPs to be identified from a randomized peptide library,which will enable the use of combinatorial protein engineer-ing methods for the discovery of novel CPPs. In this study,we used the neuronal-like PC12 cell line for selections. Inthe future, other desirable cell culture systems, including pri-mary cells, can be used for selecting CPPs. By selectinglibraries over different cell types, and performing negativeselections, this process may also be useful for discoveringCPPs that exhibit targeting or ‘‘homing’’ phenotypes. Conclusions We have developed a PD system based on a p50 DNAbinding domain for the selection of functional CPPs. Theplasmid vectors, pTAT and pCON, were constructed basedon the pRES115 vector, which was modified to contain aHis tag, a FLAG tag, and a mammalian EYFP gene to serveas a cargo for the indication of functional cellular delivery.The protein/DNA complexes were partially purified andadded to PC12 cells, but the complex with the TAT peptidein the construct did not deliver the plasmid DNA to the cells.The complex with the TAT and HA2 peptide in the con-struct, as well as the use of PEI, also did not result in trans-fected cells. The addition of Lipofectamine to the protein/ DNA complexes enabled the demonstration of TAT-medi-ated delivery of the PD system to PC12 cells. This platformrepresents a new method for the identification of peptideswith cell penetrating phenotypes, and this method is cur-rently being used in our laboratories in combinatorial experi-ments to discover new CPPs with desired features andproperties. Literature Cited 1. Dietz GP, Bahr M. Delivery of bioactive molecules into thecell: the Trojan horse approach.  Mol Cell Neurosci . 2004;27:85–131.2. Joliot A, Prochiantz A. Transduction peptides: from technologyto physiology.  Nat Cell Biol . 2004;6:189–196.3. Chauhan A, Tikoo A, Kapur AK, Singh M. The taming of thecell penetrating domain of the HIV Tat: myths and realities.  J Controlled Release . 2007;117:148–162.4. Simon MJ, Gao S, Kang WH, Banta S, Morrison B III. TAT-mediated intracellular protein delivery to primary brain cells isdependent on glycosaminoglycan expression.  Biotechnol Bioeng .2009;104:10–19.5. Heitz F, Morris MC, Divita G. Twenty years of cell-penetratingpeptides: from molecular mechanisms to therapeutics.  Br J  Pharmacol . 2009;157:195–206.6. Gao S, Simon MJ, Morrison B III, Banta S. Bifunctional chi-meric fusion proteins engineered for DNA delivery: optimiza-tion of the protein to DNA ratio.  Biochim Biophys Acta .2009;1790:198–207.7. Gupta B, Levchenko TS, Torchilin VP. Intracellular delivery of large molecules and small particles by cell-penetrating proteinsand peptides.  Adv Drug Deliv Rev . 2005;57:637–651.8. Trehin R, Merkle HP. Chances and pitfalls of cell penetratingpeptides for cellular drug delivery.  Eur J Pharm Biopharm .2004;58:209–223.9. Torchilin VP. Tat peptide-mediated intracellular delivery of pharmaceutical nanocarriers.  Adv Drug Deliv Rev . 2008;60:548– 558.10. Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo pro-tein transduction: delivery of a biologically active protein intothe mouse.  Science . 1999;285:1569–1572.11. Eguchi A, Meade BR, Chang YC, Fredrickson CT, Willert K,Puri N, Dowdy SF. Efficient siRNA delivery into primary cellsby a peptide transduction domain-dsRNA binding domain fusionprotein.  Nat Biotechnol . 2009;27:567–571.12. Kerkis A, Hayashi MA, Yamane T, Kerkis I. Properties of cellpenetrating peptides (CPPs).  IUBMB Life . 2006;58:7–13.13. Vives E. Present and future of cell-penetrating peptide mediateddelivery systems: ‘‘is the Trojan horse too wild to go only toTroy?’’.  J Controlled Release . 2005;109:77–85.14. Cai SR, Xu G, Becker-Hapak M, Ma M, Dowdy SF, McLeodHL. The kinetics and tissue distribution of protein transductionin mice.  Eur J Pharm Sci . 2006;27:311–319.15. Vives E, Schmidt J, Pelegrin A. Cell-penetrating and cell-target-ing peptides in drug delivery.  Biochim Biophys Acta .2008;1786:126–138.16. Belting M, Wittrup A. Developments in macromolecular drugdelivery.  Methods Mol Biol . 2009;480:1–10.17. Lin H, Cornish VW. Screening and selection methods for large-scale analysis of protein function.  Angew Chem Int Ed Engl .2002;41:4402–4425.18. Sergeeva A, Kolonin MG, Molldrem JJ, Pasqualini R, Arap W.Display technologies: application for the discovery of drug andgene delivery agents.  Adv Drug Deliv Rev . 2006;58:1622–1654.19. Cull MG, Miller JF, Schatz PJ. Screening for receptor ligandsusing large libraries of peptides linked to the C-terminus of theLac Repressor.  Proc Natl Acad Sci USA . 1992;89:1865–1869.20. Choi YS, Pack SP, Yoo YJ. Development of a plasmid displaysystem using GAL4 DNA binding domain for the in vitroscreening of functional proteins.  Biotechnol Lett  . 2005;27:1707– 1711.21. Speight RE, Hart DJ, Sutherland JD, Blackburn JM. A newplasmid display technology for the in vitro selection of func-tional phenotype-genotype linked proteins.  Chem Biol .2001;8:951–965.22. Gao S. Characterization of the TAT cell penetrating peptideand directed evolution of new cell penetrating peptides for pro-tein and nucleotide delivery to neuronal-like cells. PhD Thesis.New York: Columbia University; 2009.23. Hart DJ, Speight RE, Sutherland JD, Blackburn JM. Analysis of the NF-kappaB p50 dimer interface by diversity screening.  J Mol Biol . 2001;310:563–575.24. Patrick WM, Blackburn JM. In vitro selection and characteriza-tion of a stable subdomain of phosphoribosylanthranilate isom-erase.  FEBS J  . 2005;272:3684–3697.25. Wadia JS, Stan RV, Dowdy SF. Transducible TAT-HA fuso-genic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis.  Nat Med  . 2004;10:310–315.26. Sugita T, Yoshikawa T, Mukal Y, Yanianada N, Sunao IA,Nagano K, Yoshida Y, Shibata H, Yoshioka Y, Nakagawa S,Kamada H, Tsunoda SI, Tsutsumi Y. Improved cytosolic trans-location and tumor-killing activity of Tat-shepherdin conjugatesmediated by co-treatment with Tat-fused endosome-disruptiveHA2 peptide.  Biochem Biophys Res Commun . 2007;363:1027– 1032.27. Lundberg P, El-Andaloussi S, Sutlu T, Johansson H, Langel U.Delivery of short interfering RNA using endosomolytic cell-penetrating peptides.  FASEB J  . 2007;21:2664–2671.28. Rajagopalan R, Xavier J, Rangaraj N, Rao NM, Gopal V.Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu mediateefficient non-viral gene delivery.  J Gene Med  . 2007;9:275–286. Biotechnol. Prog.,  2010, Vol. 26, No. 6  1799  29. Ignatovich IA, Dizhe EB, Pavlotskaya AV, Akifiev BN, BurovSV, Orlov SV, Perevozchikov AP. Complexes of plasmid DNAwith basic domain 47–57 of the HIV-1 Tat protein are trans-ferred to mammalian cells by endocytosis-mediated pathways.  J Biol Chem . 2003;278:42625–42636.30. Liu Z, Li M, Cui D, Fei J. Macro-branched cell-penetrating peptidedesign for gene delivery.  J Controlled Release . 2005;102:699–710.31. Hashida H, Miyamoto M, Cho Y, Hida Y, Kato K, KurokawaT, Okushiba S, Kondo S, Dosaka-Akita H, Katohl H. Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a newDNA delivery tool.  Br J Cancer  . 2004;90:1252–1258.32. Xavier J, Singh S, Dean DA, Rao NM, Gopal V. Designedmulti-domain protein as a carrier of nucleic acids into cells.  J Controlled Release . 2009;133:154–160.33. Kilk K, El-Andaloussi S, Jarver P, Meikas A, Valkna A, BartfaiT, Kogerman P, Metsis M, Langel U. Evaluation of transportan10 in PEI mediated plasmid delivery assay.  J Controlled  Release . 2005;103:511–523.34. Zhang C, Yadava P, Hughes J. Polyethylenimine strategies for plasmid delivery to brain-derived cells.  Methods . 2004;33:144– 150.ManuscriptreceivedMay 20,2010. 1800  Biotechnol. Prog.,  2010, Vol. 26, No. 6
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