protein liposome conjugates using cysteine-lipids … · protein-liposome conjugates using...

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Protein-Liposome Conjugates Using Cysteine-Lipids And Native Chemical Ligation Sanne W. A. Reulen, ², | Wilco W. T. Brusselaars, ², | Sander Langereis, Willem J. M. Mulder, § Monica Breurken, ² and Maarten Merkx* Laboratory of Macromolecular and Organic Chemistry, and Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, and SyMO-Chem BV, P.O. Box 513, 5600 MB Eindhoven, The Netherlands. Received September 7, 2006; Revised Manuscript Received December 19, 2006 Liposomes have become popular drug delivery vehicles and have more recently also been applied as contrast agents for molecular imaging. Most current methods for functionalization of liposomes with targeting proteins rely on reactions of amine or thiol groups at the protein exterior, which generally result in nonspecific conjugation at multiple sites on the protein. In this study, we present native chemical ligation (NCL) as a general method to covalently couple recombinant proteins in a highly specific and chemoselective way to liposomes containing cysteine-functionalized phospholipids. A cysteine-functionalized phospholipid (Cys-PEG-DSPE) was prepared and shown to readily react with the MESNA thioester of EYFP, which was used as a model protein. Characterization of the EYFP-liposomes using fluorescence spectroscopy showed full retention of the fluorescent properties of conjugated EYFP and provides a lower limit of 120 proteins per liposome. The general applicability of NCL was further tested using CNA35, a collagen-binding protein recently applied in fluorescent imaging of collagen. NCL of CNA35 thioester yielded liposomes containing 100 copies of CNA35 per liposome. The CNA35-liposomes were shown to be fully functional and bind collagen with a 150-fold higher affinity compared to CNA35. Our results show that NCL is an attractive addition to existing conjugation methods that allows direct, covalent, and highly specific coupling of recombinant proteins to liposomes and other lipid-based assemblies. INTRODUCTION Liposomes and micelles have attracted a lot of interest as drug delivery vehicles (1, 2) and more recently as carriers of contrast agents (MRI, ultrasound) in molecular imaging (3-5). An important development in these fields has been the introduc- tion of “sterically stabilized” or “stealth” liposomes that have an increased circulation time and altered biodistributions (6). These liposomes typically contain 5% PEGylated phos- pholipids resulting in a liposome surface with low immuno- genicity and increased liposome stability (7-9). For applications in molecular imaging and targeted drug delivery, the liposomes also need to be functionalized with targeting ligands, which in most cases are proteins (e.g., antibodies) or peptides. Most current synthetic strategies to covalently couple proteins to liposomes involve either amine or thiol groups at the exterior of the protein (10, 11). Although methods for direct coupling to liposomes have been reported (12), amine groups are commonly converted to thiol groups, which are subsequently reacted with phospholipids containing thiol reactive groups such as maleimides, activated disulfides, or iodoacetyl groups (2). All of these conjugation methods are nonspecific and result in conjugation at multiple sites on the protein, which sometimes also results in protein inactivation. Due to this lack of control over the conjugation site, protein conjugation is still, to some extent, a process of trial and error. An approach to circumvent the nonspecific modification of proteins is the use of so-called docking proteins as intermediates between the liposome and the targeting protein. Examples include the use of protein G as a docking site for IgG (13) and the use of RNase I as a docking site of proteins with a C-peptide tag (14-16). Although these approaches result in a more homogeneous presentation of targeting ligands on the liposomes, they require additional conjugation steps and depend on a noncovalent bond between targeting ligand and docking protein. To date, no general strategy is available that allows the direct covalent conjugation of liposomes to a single, precisely defined site on a recombinant protein. Native chemical ligation (NCL) was first reported by Dawson et al. as a unique method to ligate two unprotected peptide fragments to form a native peptide bond, thereby allowing the complete chemical synthesis of large proteins (17, 18). NCL is a chemoselective reaction that occurs spontaneously between a peptide with a C-terminal thioester and a peptide with an N-terminal cysteine under aqueous conditions at neutral pH. The possibilities of NCL have been extended by the develop- ment of expression systems that use self-cleavable intein domains to generate recombinant proteins with C-terminal thioester groups (19). NCL has been applied to attach synthetic moieties such as fluorescent dyes, biotin, isoprenyl groups, and dendrimers to recombinant proteins, and has also been used in the site-specific immobilization of proteins to surfaces (20- 23). Here, we report on the application of NCL as a method to covalently couple recombinantly expressed proteins in a highly specific and chemoselective way to liposomes containing cysteine-functionalized phospholipids. While this work was in progress, Bertozzi and co-workers reported the novel synthesis of cysteine-functionalized phospholipids for the lipidation of green fluorescent protein (GFP) and subsequent incorporation of the lipidated GFP in supported lipid bilayers (24). The focus of our work was to explore the suitability of NCL in the * Corresponding author. E-mail: [email protected]. Fax: (+31) 40- 245-1036. ² Laboratory of Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology. SyMO-Chem BV. § Biomedical NMR, Department of Biomedical Engineering, Eind- hoven University of Technology. | Both authors contributed equally to the work presented. 590 Bioconjugate Chem. 2007, 18, 590-596 10.1021/bc0602782 CCC: $37.00 © 2007 American Chemical Society Published on Web 02/22/2007

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Page 1: Protein Liposome Conjugates Using Cysteine-Lipids … · Protein-Liposome Conjugates Using Cysteine-Lipids And ... Liposomes have become popular drug delivery vehicles and have

Protein-Liposome Conjugates Using Cysteine-Lipids And Native ChemicalLigation

Sanne W. A. Reulen,†,| Wilco W. T. Brusselaars,†,| Sander Langereis,‡ Willem J. M. Mulder,§

Monica Breurken,† and Maarten Merkx*,†

Laboratory of Macromolecular and Organic Chemistry, and Biomedical NMR, Department of Biomedical Engineering,Eindhoven University of Technology, and SyMO-Chem BV, P.O. Box 513, 5600 MB Eindhoven, The Netherlands.Received September 7, 2006; Revised Manuscript Received December 19, 2006

Liposomes have become popular drug delivery vehicles and have more recently also been applied as contrastagents for molecular imaging. Most current methods for functionalization of liposomes with targeting proteinsrely on reactions of amine or thiol groups at the protein exterior, which generally result in nonspecific conjugationat multiple sites on the protein. In this study, we present native chemical ligation (NCL) as a general method tocovalently couple recombinant proteins in a highly specific and chemoselective way to liposomes containingcysteine-functionalized phospholipids. A cysteine-functionalized phospholipid (Cys-PEG-DSPE) was preparedand shown to readily react with the MESNA thioester of EYFP, which was used as a model protein. Characterizationof the EYFP-liposomes using fluorescence spectroscopy showed full retention of the fluorescent properties ofconjugated EYFP and provides a lower limit of 120 proteins per liposome. The general applicability of NCL wasfurther tested using CNA35, a collagen-binding protein recently applied in fluorescent imaging of collagen. NCLof CNA35 thioester yielded liposomes containing∼100 copies of CNA35 per liposome. The CNA35-liposomeswere shown to be fully functional and bind collagen with a 150-fold higher affinity compared to CNA35. Ourresults show that NCL is an attractive addition to existing conjugation methods that allows direct, covalent, andhighly specific coupling of recombinant proteins to liposomes and other lipid-based assemblies.

INTRODUCTION

Liposomes and micelles have attracted a lot of interest asdrug delivery vehicles (1, 2) and more recently as carriers ofcontrast agents (MRI, ultrasound) in molecular imaging (3-5).An important development in these fields has been the introduc-tion of “sterically stabilized” or “stealth” liposomes that havean increased circulation time and altered biodistributions (6).These liposomes typically contain∼5% PEGylated phos-pholipids resulting in a liposome surface with low immuno-genicity and increased liposome stability (7-9). For applicationsin molecular imaging and targeted drug delivery, the liposomesalso need to be functionalized with targeting ligands, which inmost cases are proteins (e.g., antibodies) or peptides. Mostcurrent synthetic strategies to covalently couple proteins toliposomes involve either amine or thiol groups at the exteriorof the protein (10, 11). Although methods for direct couplingto liposomes have been reported (12), amine groups arecommonly converted to thiol groups, which are subsequentlyreacted with phospholipids containing thiol reactive groups suchas maleimides, activated disulfides, or iodoacetyl groups (2).All of these conjugation methods are nonspecific and result inconjugation at multiple sites on the protein, which sometimesalso results in protein inactivation. Due to this lack of controlover the conjugation site, protein conjugation is still, to someextent, a process of trial and error. An approach to circumventthe nonspecific modification of proteins is the use of so-called

docking proteins as intermediates between the liposome and thetargeting protein. Examples include the use of protein G as adocking site for IgG (13) and the use of RNase I as a dockingsite of proteins with a C-peptide tag (14-16). Although theseapproaches result in a more homogeneous presentation oftargeting ligands on the liposomes, they require additionalconjugation steps and depend on a noncovalent bond betweentargeting ligand and docking protein. To date, no general strategyis available that allows the direct covalent conjugation ofliposomes to a single, precisely defined site on a recombinantprotein.

Native chemical ligation (NCL) was first reported by Dawsonet al. as a unique method to ligate two unprotected peptidefragments to form a native peptide bond, thereby allowing thecomplete chemical synthesis of large proteins (17, 18). NCL isa chemoselective reaction that occurs spontaneously between apeptide with a C-terminal thioester and a peptide with anN-terminal cysteine under aqueous conditions at neutral pH.The possibilities of NCL have been extended by the develop-ment of expression systems that use self-cleavable inteindomains to generate recombinant proteins with C-terminalthioester groups (19). NCL has been applied to attach syntheticmoieties such as fluorescent dyes, biotin, isoprenyl groups, anddendrimers to recombinant proteins, and has also been used inthe site-specific immobilization of proteins to surfaces (20-23). Here, we report on the application of NCL as a method tocovalently couple recombinantly expressed proteins in a highlyspecific and chemoselective way to liposomes containingcysteine-functionalized phospholipids. While this work was inprogress, Bertozzi and co-workers reported the novel synthesisof cysteine-functionalized phospholipids for the lipidation ofgreen fluorescent protein (GFP) and subsequent incorporationof the lipidated GFP in supported lipid bilayers (24). The focusof our work was to explore the suitability of NCL in the

* Corresponding author. E-mail: [email protected]. Fax: (+31) 40-245-1036.

† Laboratory of Macromolecular and Organic Chemistry, Departmentof Biomedical Engineering, Eindhoven University of Technology.

‡ SyMO-Chem BV.§ Biomedical NMR, Department of Biomedical Engineering, Eind-

hoven University of Technology.| Both authors contributed equally to the work presented.

590 Bioconjugate Chem. 2007, 18, 590−596

10.1021/bc0602782 CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 02/22/2007

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synthesis of protein-liposome conjugates, however, demon-strating for the first time that protein thioesters can be directlyconjugated to cysteine-functionalized phospholipids embeddedin liposomes.

EXPERIMENTAL PROCEDURESGeneral.Unless stated otherwise, all reagents and chemicals

were obtained from commercial sources and used without furtherpurification. Dichloromethane (DCM) was obtained by distil-lation from P2O5. 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[amino(poly(ethylene glycol))2000] (NH2-PEG-DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(poly(ethylene glycol))2000] (PEG-DSPE), and 1,2-dipalmi-toyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamineB sulfonyl) (rhodamine-DPPE) were purchased from AvantiPolar Lipids (Albaster, U.S.A.). Gd-DTPA-bis(stearylamide)was purchased from Gateway Chemical Technology (St. Louis,MO). Trityl-protected cysteine (Tr-Cys(Tr)-OH) was obtainedfrom Bachem (Bubendorf, Switzerland). Trityl-protected suc-cinimidyl-activated cysteine (Tr-Cys(Tr)-OSu) was preparedaccording to a literature procedure (20). UV-vis spectra wererecorded on a Shimadzu Multispec 1501 spectrophotometer.Fluorescence spectra were obtained on an Edinburgh InstrumentsFS920 double-monochromator spectrophotometer. Primers usedfor all the cloning procedures were supplied by MWG (Ebers-berg, Germany).

Synthesis of Cysteine-Functionalized 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly(ethylene gly-col))2000] (Cys-PEG-DSPE). NH2-PEG-DSPE (1) (100mg, 35.8 µmol) was dissolved in DCM (1 mL) under anatmosphere of argon. Tr-Cys(Tr)-OSu (30 mg, 43µmol) andtriethylamine (10µL, 71 µmol) were added to the solution. Thereaction proceeded overnight at room temperature. The solutionwas concentrated under reduced pressure, and the crude productwas dissolved in CHCl3 (0.5 mL). The crude product waspurified by column chromatography (silica, CHCl3/MeOH, 19:1v/v f 9:1 v/v), and trityl-protected Cys-PEG-DSPE (2) (44mg, 13 µmol) was obtained in 36% yield. Compound2 wasdissolved in triethyl silane (25µL, 158 mmol). Subsequently,a solution of trifluoroacetic acid (1 mL) and DCM (1 mL) wasadded. The obtained solution was stirred for 2 h at roomtemperature. The solution was concentrated under reducedpressure, and the crude product was precipitated in diethyl ether.The product was filtrated and dried under reduced pressure togive Cys-PEG-DSPE lipid (3) (34 mg, 12 µmol, 92%).Compounds1-3 were analyzed in detail with RP-HPLC andMALDI-TOF (see Supporting Information).

Plasmid Constructs.The EYFP gene was amplified by PCRfrom vector pEYFP-N1 (Clontech) using the primers 5′-GTGGTC ATA TGG TGA GCA AGG GCG AG-3′ and 5′-GTGGTG AAT TCC TTG TAC AGC TCG TCC ATG C-3′. TheCNA35 gene was amplified from pQE30CNA35 (a kind giftfrom Dr. Magnus Ho¨ok, Texas A & M University, U.S.A. (25))using the primers 5′-GTG GTC ATA TGG GAT CCG CACGAG ATA TTT C-3′ and 5′-GTG GTT GCT CTT CCG CATGCC TTG GTA TCT TTA TCC TGT TTT AAA AC-3′. ThePCR products and the pTXB1 vector (IMPACT system, NewEngland Biolabs) were double-digested with the restrictionendonucleasesNde I and SapI (CNA35) or Nde I and EcoR I(EYFP) followed by ligation of the amplified DNA fragmentsin the open plasmids to yield pTXB1-CNA35 and pTXB1-EYFP, respectively. DNA sequencing using T7 promoter andintein-specific reversed primers (New England Biolabs) con-firmed the correct in-frame fusion of the proteins with the inteinsequence.

Protein Expression and Purification.The expression plas-mids pTXB1-CNA35 and pTXB1-EYFP were transformed

into E. coli BL21 (DE3) cells. The same expression conditionswere used for both proteins. Bacteria were grown in LB mediumcontaining 100µg/mL ampicillin at 37°C and 250 rpm to anoptical density (OD600nm) between 0.6 and 0.8. Protein expres-sion was induced with 0.5 mM IPTG, and the cultures wereincubated overnight at 15°C. Cells were harvested by centrifu-gation for 30 min at 8000g at 4 °C. The supernatant wasremoved, and the cell pellet was resuspended using theBugBuster protocol (Novagen). After incubation for 20 min atroom temperature, the cell suspension was centrifuged at 16 000g for 20 min at 4°C. The supernatant was directly applied to acolumn of chitin beads and equilibrated with 10 column volumesof column buffer (20 mM sodium phosphate, 0.1 mM EDTA,0.5 M NaCl, pH 8). The column was washed with 10 volumesof column buffer after which the column was quickly flushedwith 3 column volumes of cleavage buffer (20 mM sodiumphosphate, 0.1 mM EDTA, 0.5 M NaCl, pH 6) containing 50mM sodium 2-mercaptoethanesulfonate (MESNA) and incu-bated overnight at room temperature. Elution fractions werecollected and pooled, after which the cleavage step was repeatedto gain more thioester-terminated proteins. The proteins werebuffer-exchanged into 10 mM HEPES, 135 mM NaCl, pH 8.0(HBS) using Amicon ultracentrifuge tubes (MWCO 10 kDa).The concentrations of EYFP protein with a C-terminal MESNAthioester (EYFP-MESNA) and CNA35 protein with a C-terminal MESNA thioester (CNA35-MESNA) were determinedby UV-vis usingε514nm) 84 000 M-1 cm-1 (26) andε280nm)33 167 M-1 cm-1, respectively. 1 LE. coli culture typicallyyielded 20 mg of EYFP-MESNA and 40 mg of CNA35-MESNA.

Ligation of EYFP to Pure Cysteine-Functionalized Phos-pholipids. Cys-PEG-DSPE lipid 3 was dissolved in buffercontaining 200 mM sodium phosphate, 200 mM NaCl, pH 7.2.Native chemical ligation reactions were performed in 200µLwith a final concentration of EYFP-COSR of 87µM. A 10-fold molar excess of compound3 (final concentration 870µM)was used. 1% (v/v) benzyl mercaptan and 1% (v/v) thiophenol,or 100 mM MESNA, were added to the ligation mixtures. Afterovernight incubation at room temperature, the samples werecentrifuged to remove precipitate, and the supernatant wasanalyzed using SDS-PAGE.

Liposome Preparation.Liposomes were prepared by lipidfilm hydration as described previously (27). A mixture of DSPC(37µmol), Gd-DTPA-bis(stearylamide) (25µmol), cholesterol(33 µmol), PEG-DSPE (2.5µmol), rhodamine-DPPE (0.1µmol), and Cys-PEG-DSPE (2.5µmol) was dissolved inCHCl3/MeOH 1:1 (v/v) and concentrated under reduced pressureat room temperature. The obtained lipid film was hydrated inHBS buffer (4 mL). This dispersion was extruded five times at65 °C through polycarbonate membrane filters with pores of100 or 200 nm. Phospholipid concentrations were determinedby phosphate analysis according to Rouser (28). The amountof lipids per liposome was calculated using a lipid surface areaof 0.6 nm2 (29) and assuming unilamellar liposomes.

Ligation of EYFP to Cysteine-Liposomes.Native chemicalligation of cysteine-liposomes (450µM Cys-PEG-DSPE)with EYFP-MESNA (33µM) was performed for 48 h at 20°Cin HBS, pH 8, containing either 100 mM MESNA or 1% (v/v)thiophenol and 1% (v/v) benzyl mercaptan. The couplingefficiency was monitored via SDS-PAGE analysis. The amountof lipidated EYFP was calculated by scanning of the SDS-PAGE gel and integration of the bands of unreacted and reactedprotein. The liposome concentration was calculated from thelipid concentration that was determined via phosphate analysisusing the assumption of unilamellar liposomes. The number ofEYFP proteins per liposome was then obtained by dividing theconcentration of lipidated EYFP by the liposome concentration.

Technical Notes Bioconjugate Chem., Vol. 18, No. 2, 2007 591

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The EYFP-liposomes were ultracentrifuged in a KontronCentrikon T-2060 ultracentrifuge with a TFT 70.38 rotor for1 h at 270 000g and 20°C, after which the liposomal pelletwas resuspended in HBS. Pellet and supernatant were analyzedusing SDS-PAGE to confirm the separation between reactedand unreacted protein. The incorporation of EYFP was alsoestablished using fluorescence spectroscopy. Emission spectraof EYFP-liposomes were measured using an excitation wave-length of 490 nm. The ratio of the EYFP (527 nm) andrhodamine (590 nm) peaks in the emission spectrum wascompared to a calibration curve with known molar ratios ofEYFP to rhodamine, thus providing an alternative method todetermine the amount of EYFP per liposome.

Ligation of CNA35 to Cysteine-Liposomes.Native chemi-cal ligation of cysteine-liposomes (180µM Cys-PEG-DSPE)with CNA35 thioester (50µM) was performed for 24 h at 20°Cin HBS, pH 8, containing 100 mM MESNA. The amount oflipidated CNA35 was calculated by scanning the SDS-PAGEgel and integrating the bands of unreacted and reacted protein.The liposome concentration was calculated from the lipidconcentration that was determined via phosphate analysis usingthe assumption of unilamellar liposomes. The amount oflipidated CNA35 was divided over the liposome concentrationto determine the number of CNA35 proteins per liposome.Ultracentrifugation of CNA35-liposomes was performed in aKontron Centrikon T-2060 ultracentrifuge with a TFT 70.38rotor for 1 h at 270 000g and 20°C. The obtained liposomalpellet was resuspended in HBS. Pellet and supernatant wereanalyzed using SDS-PAGE to confirm the separation betweenreacted and unreacted protein. The concentration of CNA35 inthe liposome fraction was determined with the Quant-iT ProteinAssay Kit (Invitrogen) according to manufacturer instructions.This amount of lipidated CNA35 was divided by the liposomeconcentration to determine the number of CNA35 proteins perliposome.

Collagen Binding Assay.96 well Corning EIA/RIA micro-plates were coated overnight at 4°C with 1.4µg/well (45 µL)rat tail collagen type I (Sigma, C7661) in TBS (50 mM Tris,150 mM NaCl, pH 7.5). After overnight incubation, the plateswere blocked with 100µL TBS containing 5% (w/v) skim milkpowder for 2 h atroom temperature. After washing the plates3 times with 300µL TBS, the plates were incubated withCNA35-liposomes or nonmodified liposomes in HBS supple-mented with 5% (w/v) skim milk powder for 3 h at roomtemperature. Plates were washed 5 times with 50 mM Tris, 500mM NaCl, pH 7.5, and subsequently washed 2 times with TBS.The fluorescence of the rhodamine-containing liposomes wasmeasured at 620 nm in triplicate on a Thermo Fluoroskan AscentFL plate reader after excitation at 578 nm.

RESULTS AND DISCUSSIONIn order to use native chemical ligation as a site-specific

method to prepare protein-liposomes, the PEG terminus ofPEG-DSPE was functionalized with a cysteine containing afree amine group. The introduction of the reactive group at theend of the PEG chain (far removed from the lipid tail) has beenreported to enhance ligation efficiencies (30) and was alsoapplied in previous work using maleimide-functionalized phos-pholipids (27, 31). Cys-PEG-DSPE (3) was obtained in atwo-step synthesis starting from the commercially available1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(po-ly(ethylene glycol))2000] (1) (Scheme 1). In the first step, theamine end group of1 was reacted with succinimidyl-activatedtrityl-protected cysteine to yield intermediate2 in 36% yield.The second step involved deprotection of the cysteine usingtrifluoroacetic acid (TFA) to give Cys-PEG-DSPE3 in 92%yield, which was confirmed using RP-HPLC and MALDI-TOF(see Supporting Information).

Enhanced yellow fluorescent protein (EYFP) was chosen asa model protein to study the performance of Cys-PEG-DSPEin native chemical ligation reactions. The fluorescence of EYFPdepends on proper folding of the protein and can thus be usedto assess protein stability after ligation. In addition, fluorescencecan be used to determine the amount of proteins conjugatedper liposome. A C-terminal fusion protein of EYFP with inteinand chitin binding domains was expressed inE. coli using theIMPACT system. The fusion protein was purified on a chitin resinand treated with 50 mM MESNA to induce the intein-catalyzedcleavage of the fusion protein and the formation of EYFP witha C-terminal MESNA thioester (EYFP-MESNA) (Scheme 2).SDS-PAGE and ESI-MS analysis (see Supporting Information)showed the presence of a single protein with a mass of 27 849Da that corresponds to the calculated mass of EYFP-MESNA(theoretical mass: 27845.2 Da). We first studied the reactionof pure Cys-PEG-DSPE (not incorporated in a liposome) withEYFP-MESNA in the presence of two different thiol catalysts,thiophenol/benzyl mercaptan and MESNA. Since ligation ofCys-PEG-DSPE to EYFP increases its molecular weight byapproximately 3 kDa, the ligation reaction could be monitored

Scheme 1. Synthesis of Cys-PEG-DSPE (3)a

a (a) DCM, triethylamine, Tr-Cys(Tr)-OSu; (b) triethylsilane, TFA,DCM.

Scheme 2. Synthesis of Thioester-Terminated Proteins UsingSelf-Cleavable Intein Domains

592 Bioconjugate Chem., Vol. 18, No. 2, 2007 Reulen et al.

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using SDS-PAGE. Nearly quantitative reaction of EYFP to thelipidated form of EYFP (EYFP-PEG-DSPE) was observedin the presence of thiophenol, whereas a much lower conversion(∼5%) was obtained using MESNA as the catalyst (Figure 1).Since PEGylated phospholipids such as Cys-PEG-DSPE areknown to form relatively stable micelles (32), the ligationreaction probably resulted in the formation of EYFP micelles.We are currently characterizing the properties of these putativeprotein micelles.

Having established the suitability of Cys-PEG-DSPE innative chemical ligation reactions, we next tested whether thesame native chemical ligation can also be performed when Cys-PEG-DSPE is incorporated into liposomes. Liposomes contain-ing 2.5% of Cys-PEG-DSPE, Gd-DTPA-bis(stearylamide)lipids, and rhodamine-functionalized lipids were prepared bylipid film hydration as described previously (27). We againcompared thiophenol/benzyl mercaptan and MESNA as catalysts

and monitored the reaction using SDS-PAGE. Figure 2 clearlyshows the presence of lipidated EYFP proteins upon reactionof 33 µM EYFP-thioester with 450µM liposome-embeddedCys-PEG-DSPE. To the best of our knowledge, this is thefirst report that describes the direct coupling of proteins toliposomes via native chemical ligation. Although the ligationreaction is more efficient in the presence of thiophenol/benzylmercaptan (conversion≈ 30%) than in the presence of MESNA(conversion ≈ 10%), the use of MESNA has importantadvantages, in particular, when working with lipid-basedsystems. Thiophenol and benzyl mercaptan are poorly watersoluble and are likely to accumulate in the phospholipid bilayerof liposomes, making it almost impossible to remove these toxiccompounds after the ligation reaction. MESNA is a water-soluble thiol with a nonoffensive odor compared to thiophenoland benzyl mercaptan, and is easily removed after ligation viacentrifugation.

Figure 1. Native chemical ligation of Cys-PEG-DSPE3 (870µM) with EYFP-MESNA (87µM) in 0.2 M sodium phosphate pH 7.2 with either100 mM MESNA or 1% (v/v) thiophenol+ 1% benzyl mercaptan (THIOPHENOL).

Figure 2. Native chemical ligation of liposomes containing Cys-PEG-DSPE (450µM cysteine-lipid) with EYFP-MESNA (33 µM) in HBSpH 8 with either 100 mM MESNA or 1% (v/v) thiophenol+ 1% benzyl mercaptan (THIOPHENOL).

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To prove that the lipidated EYFP observed in SDS-PAGEwas still attached to the liposomes, liposomes were separatedfrom unreacted EYFP via ultracentrifugation. SDS-PAGEanalysis showed that all of the unreacted EYFP was present inthe supernatant, whereas most of the lipidated EYFP was foundassociated with the liposomal pellet (see Supporting Informa-tion). Fluorescence spectroscopy was used to determine thenumber and proper folding of the conjugated EYFP proteins.The fluorescence emission spectrum of the EYFP-liposomesshowed peaks at 527 and 590 nm, corresponding to EYFP andrhodamine, respectively (Figure 3). The observation of EYFPfluorescence not only demonstrates that the protein kept itsnative fold after ligation, but the relative intensity of the EYFPfluorescence compared to the rhodamine fluorescence can alsobe used to estimate the number of EYFP proteins per liposome.The ratio of EYFP to rhodamine was compared to a calibrationcurve with known molar ratios of EYFP to rhodamine, yieldingapproximately 120 proteins per 200 nm liposome. Anothermethod to estimate the number of proteins per liposome is tocalculate the amount of lipidated protein from the relativeintensities of lipidated and non-lipidated protein bands on theSDS-PAGE gel obtained after NCL, and divide this number bythe original liposome concentration. This calculation assumesthat all lipidated protein is incorporated into liposomes andyielded 180 EYFP per 200 nm liposome.

Although EYFP is a good model protein to prove that proteinscan be conjugated to liposomes via native chemical ligation,we also wanted to use a biomedically relevant protein with aspecific binding function. We recently developed a collagen-specific fluorescent probe based on a collagen-binding proteindomain (CNA35) from the bacterial adhesion protein ofStaphylococcus aureus(33). Solid-phase binding assays showeda dissociation constant of approximately 0.5µM for collagentype I. In addition to providing an example of a targetedliposomal contrast agent, we were also interested to see whetherattachment of multiple CNA35 proteins to a liposomal scaffoldwould yield a probe with a higher affinity for collagen due tomultivalent interactions between CNA35 and collagen.

The IMPACT expression vector pTXB1 was again used toobtain recombinant CNA35 with a C-terminal MESNA thioester(Scheme 2). An alanine residue was added to the C-terminusof CNA35 during cloning, as this residue is known to enhancethe rate of native chemical ligation reactions (18). Affinitypurification on a chitin column and subsequent cleavage withMESNA yielded CNA35-MESNA in excellent yield. SDS-PAGE analysis typically showed the presence of several smallerbands besides the major band at 35 kDa, which are probablydegradation products of CNA35 (Figure 4). ESI-MS showed a

single protein peak with a mass of 34 757 Da (theoreticalmass: 34774 Da). Collagen-binding assays using fluorescentlylabeled CNA35-MESNA showed that the modified C-terminusdid not affect the collagen-binding properties.

CNA35-MESNA was ligated to liposomes using the sameprocedure as described for EYFP using MESNA as a catalyst.SDS-PAGE analysis again indicated the formation of lipidatedprotein by the appearance of a new band at approximately 38kDa (Figure 4). CNA35-modified liposomes were separatedfrom MESNA and unreacted CNA35 by two ultracentrifugationsteps (see Supporting Information). A protein quantificationassay was used to determine the amount of protein in theliposome fraction. This protein concentration was divided bythe known liposome concentration, yielding∼100 proteins per100 nm liposome. This number is in reasonable agreement withthe amount of lipidated CNA35 that was detected by SDS-PAGEanalysis immediately after ligation, which gave∼80 CNA35proteins per 100 nm liposome. The rhodamine fluorescence ofthe CNA35-functionalized liposomes was used to study theirbinding to a collagen-coated 96 well plate. Figure 5 showsspecific binding of these liposomes to rat tail collagen type I at

Figure 3. Emission spectrum of EYFP-liposome conjugates usingan excitation wavelength of 490 nm (solid line). The emission spectraof EYFP-MESNA (dashed line) and nonmodified rhodamine-contain-ing liposomes (dotted line) are also shown for comparison.

Figure 4. SDS-PAGE analysis of CNA35-MESNA before and afternative chemical ligation to liposomes containing Cys-PEG-DSPE.

Figure 5. Solid-phase binding assay of CNA35-liposomes to rat tailcollagen type I (solid squares). Liposome binding was monitored bymeasuring the fluorescence of the rhodamine lipids at 620 nm usingan excitation of 578 nm. Control experiments using nonmodifiedliposomes incubated on rat tail collagen type I (open triangle) andCNA35-liposomes incubated on milk-powder blocked well withoutcollagen (open squares) are also shown for comparison. The solid linerepresents a fit to a 1:1 binding model using aKd of 3 nM.

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low nanomolar concentrations. Control experiments usingnonfunctionalized liposomes or plates without collagen did notshow a similar signal. A fit of the binding curve using a simple1:1 binding model yielded aKd of 3 ( 1 nM, representing a150-fold increase in affinity compared to the protein itself. Theseresults show that CNA35 conjugated to liposomes via nativechemical ligation is fully active in binding collagen. Whetherthe observed increase in collagen affinity is due to multiple,simultaneous interactions between probe and collagen or mainlya statistical effect due to the presence of∼100 copies of proteinper liposome remains to be investigated.

In summary, we have demonstrated that native chemicalligation is an attractive method to directly conjugate recombi-nantly expressed proteins to sterically stabilized poly(ethyleneglycol) liposomes via cysteine-functionalized phospholipids. Incontrast to other conjugation methods such as thiol-maleimidereactions, NCL is highly specific and exclusively occurs at theprotein’s C-terminus, which in most proteins is not importantfor binding activity. An additional advantage of NCL is thatonly a single site in the protein is available for conjugation,whereas cross-linking is sometimes observed between liposomesand proteins using classical thiol/amine chemistries (34). Thestrategy reported here for liposomes should be readily applicableto other lipid-based imaging agents such as immunomicelles(35), iron oxide nanoparticles (31, 36), and quantum dots witha lipid coating (37-40).

ACKNOWLEDGMENT

This study was funded by the BSIK program entitledMolecular Imaging of Ischemic Heart Disease (project numberBSIK03033). The authors thank Anouk Dirksen for help withthe synthesis of the cysteine-functionalized lipid, MariekeRensen for constructing pTXB1-EYFP, Ingrid van Baal forsupplying trityl-protected succinimidyl-activated cysteine, ErikSanders for providing assistance with liposome preparation andcharacterization, and Bert Meijer, Klaas Nicolay, and GustavStrijkers for general support.

Supporting Information Available: Experimental details in-cluding the RP-HPLC and MALDI-TOF analysis of thesynthesized phospholipids, ESI-MS spectra of EYFP-MESNAand CNA35-MESNA, and SDS-PAGE analysis of the purifica-tion of EYFP-MESNA, CNA35-MESNA, EYFP-liposomes, andCNA35-liposomes. This material is available free of charge viathe Internet at http://pubs.acs.org/BC.

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