Site-Specific Albumination as an Alternative to PEGylation for the

Apr 6, 2016 - (1d) The purified sfGFP-WT and sfGFP-AzF were analyzed by SDS-PAGE to check their purity and AzF incorporation. ..... R.; Tolmachev , V...
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Site-Specific Albumination as an Alternative to PEGylation for the Enhanced Serum Half-Life in Vivo Byungseop Yang,† Sung In Lim,‡ Jong Chul Kim,† Giyoong Tae,† and Inchan Kwon*,†,‡ †

School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 61005, Republic of Korea ‡ Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22904, United States ABSTRACT: Polyethylene glycol (PEG) has been widely used as a serum half-life extender of therapeutic proteins. However, due to immune responses and low degradability of PEG, developing serum half-life extender alternatives to PEG is required. Human serum albumin (HSA) has several beneficial features as a serum half-life extender, including a very long serum half-life, good degradability, and low immune responses. In order to further evaluate the efficacy of HSA, we compared the extent of serum half-life extension of a target protein, superfolder green fluorescent protein (sfGFP), upon HSA conjugation with PEG conjugation side-by-side. Combination of site-specific incorporation of p-azido-L-phenylalanine into sfGFP and copper-free click chemistry achieved the site-specific conjugation of a single HSA, 20 kDa PEG, or 30 kDa PEG to sfGFP. These sfGFP conjugates exhibited the fluorescence comparable to or even greater than that of wild-type sfGFP (sfGFPWT). In mice, HSA-conjugation to sfGFP extended the serum half-life 9.0 times compared to that of unmodified sfGFP, which is comparable to those of PEG-conjugated sfGFPs (7.3 times for 20 kDa PEG and 9.5 times for 30 kDa PEG). These results clearly demonstrated that HSA was as effective as PEG in extending the serum half-life of a target protein. Therefore, with the additional favorable features, HSA is a good serum half-life extender of a (therapeutic) protein as an alternative to PEG.

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INTRODUCTION One of the major issues in therapeutic protein development is overcoming fast clearance of therapeutic proteins administered to patients. Researchers have put a great deal of effort into developing various serum half-life extenders by binding or conjugating them to therapeutic proteins.1 Such extended serum half-life of a therapeutic protein would improve patients’ quality of life and compliance by requiring fewer administrations. Conjugation of polyethylene glycol (PEG; PEGylation) to therapeutic proteins/peptides has been widely used to improve the serum half-life in vivo, because PEG molecules are United States Food and Drug Administration (FDA)-approved, flexible, water-soluble, and nontoxic.2 However, recent findings raised a concern regarding the immunogenicity of PEG molecules,3 which likely restricts repeated use of PEGylated therapeutics. It was reported that PEG molecules conjugated to therapeutic urate oxidase generated antibodies against PEG molecules.4 Low degradability of PEG is also a drawback. Therefore, it is clearly important to develop alternative strategies to PEGylation for serum half-life extension of therapeutic proteins. Since human serum albumin (HSA) has low immunogenicity and good degradability as well as a unusually long serum half-life (over 2 weeks), HSA recently attracted much attention as a half-life extender of therapeutics.1b,c,5 Such a long serum half-life of HSA in the human body © 2016 American Chemical Society

is attributed to increased hydrodynamic volume, electrostatic repulsion in kidneys, and FcRn-mediated recycling.5c,6 Genetic fusion or chemical conjugation of HSA (albumination) to therapeutic peptides and small-sized proteins successfully enhanced the serum half-life in vivo.7 However, each albumination technique has some limitations. In the case of genetic fusion, genetic fusion of HSA is limited to either N- or C-terminus of a target protein, and so it is not ideal to apply it to therapeutic proteins of which N- or C-terminus is critical for their therapeutic activities. As for chemical conjugation of HSA, conjugation has often been performed at lysine or cysteine residues. Since there are usually multiple reactive residues (lysines or cysteins) exposed to surface of a therapeutic protein, the conventional chemical conjugation of albumin to a therapeutic protein generates heterogeneous mixtures of the conjugates, resulting in a significant therapeutic activity loss and complicated downstream process.5c Under mild conditions, HSA is considered not aggregation-prone. Therefore, HSA has been used as a stabilizer for numerous therapeutic proteins.8 Such stabilization of therapeutic proteins is partially attributed to direct interactions between HSA and therapeutic proteins.9 Received: February 16, 2016 Revised: April 4, 2016 Published: April 6, 2016 1811

DOI: 10.1021/acs.biomac.6b00238 Biomacromolecules 2016, 17, 1811−1817

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carboxyrhodamine 110 (DBCO-PEG4-carboxyrhodamine) were purchased from Click Chemistry Tools LLC (Scottsdale, AZ). Coumarinazide was obtained from Glen Research (Sterling, VA). Disposable PD-10 Desalting columns, HiTrap Q HP anion exchange columns, HiTrap SP HP cation exchange columns, and Superdex 200 10/300 GL size exclusion columns were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, U.K.). Vivaspin centrifugal concentrators with a MWCO of 10 kDa were purchased from Sartorius (Weender Landstr, Goettingen, Germany). Human serum albumin was obtained from Sigma-Aldrich (St. Louis, MO). A GFP-specific ELISA kit was purchased from Cell Biolabs, Inc. (San Diego, CA). All other chemicals were purchased from Sigma-Aldrich and used without further purification unless otherwise indicated. Expression and Purification of sfGFP-AzF and Wild-Type sfGFP. A plasmid pEVOL-pAzF (Plasmid ID: 31186), obtained from Addgene (Cambridge, MA), encodes engineered orthogonal pair genes of tyrosyl-tRNA synthetase/amber suppressor tRNA derived from Methanococcus jannaschii, which specifically recognize AzF.1d,11 A plasmid pQE80-sfGFP_215amb encodes a sfGFP variant gene containing an amber codon at position 215.1d In order to sitespecifically incorporate AzF into sfGFP, C321delA.exp Escherichia coli host cells12 were transformed with two plasmids pEVOL-pAzF and pQE80-sfGFP_215amb to generate C321delA.exp [pEVOL-pAzF][pQE80-sfGFP_215amb] cells. C321delA.exp [pEVOL-pAzF][pQE80-sfGFP_215amb] cells were inoculated into a 2xYT medium containing ampicillin (100 μg/mL) and chloramphenicol (35 μg/mL), and were cultured at 37 °C in a shaking incubator (220 rpm). When the OD600 of 0.4 was reached, AzF solution was added to a final concentration of 1 mM. At OD600 of 0.6, protein expression was induced by adding IPTG and L-(+)-arabinose to final concentrations of 1 mM and 0.2%, respectively. After 7 h, cells were harvested by centrifugation at 12 000 rpm at 4 °C for 20 min. In order to purify sfGFP-AzF, cell pellets were resuspended with a lysis buffer containing 50 mM sodium phosphate, 300 mM sodium chloride, and 10 mM imidazole at pH 7.5. The resuspended cells were then incubated on ice for 30 min with lysozyme (200 μg/mL) and were sonicated on ice (10 s pulse and 20 s pause) for a total of 6 min. The cell lysate was centrifuged at 12 000 rpm at 4 °C for 30 min. The clear supernatant was mixed with Ni-NTA agarose and shaken at 220 rpm for 1 h on a rotary shaker at room temperature. The lysate-Ni-NTA mixture was then loaded into a column. The column was washed with a wash buffer containing 50 mM sodium phosphate, 300 mM sodium chloride, and 20 mM imidazole (pH 7.5). The sfGFP-AzF was eluted with an elution buffer containing 50 mM sodium phosphate, 300 mM sodium chloride, and 250 mM imidazole (pH 7.5). Expression and purification of wild-type sfGFP (sfGFP-WT) was carried out similarly, with the exception of using the Top10 E. coli cells transformed with pQE80sfGFP plasmid1d and inducing sfGFP expression at the OD600 of 0.9 without adding an AzF solution and L-(+)-arabinose. Spectrometric Quantification of sfGFP Variants and DyeLabeling. The molar absorbance of AzF at 280 nm was reported to be 2620 M−1 cm−1.1c The molar absorption coefficients, ε280 (M−1 cm−1), of sfGFP-WT and sfGFP-AzF, calculated according to the method in the literature,13 were 19 035 and 21 655 M−1 cm−1, respectively. The protein concentrations were obtained according to the Beer−Lambert law14 by measuring molar absorbance using a Synergy H1 four multimode microplate reader (BioTek, Winooski, VT). Incorporation of AzF into sfGFP was determined by conjugating a fluorescent dye to the azide functional group of sfGFP. The purified sfGFP-AzF was mixed with DBCO-PEG4-carboxyrhodamine 110 dye at molar ratio 1:4 in PBS (pH 7.0). The SPAAC (strain promoted azide−alkyne cycloaddition) reaction was carried out at room temperature for 2 h. The reaction solution was boiled for 10 min to eliminate intrinsic sfGFP fluorescence, and then loaded onto a protein gel to measure ingel fluorescence using ChemiDoc XRS+ Bio-Rad Laboratories Inc. (Berkeley, CA). Upon illumination at λex = 302 nm, the emitted light between 510 and 610 nm was captured. The protein gel was also stained with Coomassie blue to visualize proteins. LC-MS Analysis of sfGFP-WT and sfGFP-AzF. Molecular masses of sfGFP-WT and sfGFP-AzF were measured by liquid chromatog-

However, during HSA conjugation, there might be some perturbation of HSA structure leading to aggregation. Considering the potential of unwanted immune responses caused by protein aggregation,10 stability of HSA-conjugated therapeutic proteins should be carefully examined. We recently demonstrated that site-specific albumin-conjugation to a permissive site of a therapeutic protein urate oxidase can enhance the serum half-life in vivo without compromising its therapeutic activity.1c In order to evaluate the effectiveness of HSA as a serum halflife extender alternative to PEG, it is required to compare serum half-life extension of the same protein upon albumination and PEGylation side-by-side. Furthermore, either HSA or PEG should be conjugated to the same site of a target protein. To our knowledge, these were not yet extensively investigated. Therefore, in this study, we conjugated a single HSA or a single PEG molecule (20-kDa or 30-kDa) at the same site of a target protein using site-specific incorporation of a clickable nonnatural amino acid and copper-free click chemistry. Then, we compared the serum half-lives of HSA- and PEG-conjugated proteins (Figure 1). As a model protein, we chose superfolder

Figure 1. Schematic diagrams of site-specific conjugation of human serum albumin or poly(ethylene glycol) molecules to sfGFP to prepare three different sfGFP conjugates. sfGFP-AzF: sfGFP variant containing p-azido-L-phenylalanine at the 215th position; DBCO-PEG30: 30 kDa dibenzocyclooctyne-methoxy poly(ethylene glycol); DBCO-PEG20: 20 kDa dibenzocyclooctyne-methoxy poly(ethylene glycol); DBCOHSA: human serum albumin conjugated to dibenzocylooctyne-PEG4maleimide linker; sfGFP-PEG20: sfGFP-PEG30, and sfGFP-HSA: sfGFP-AzF variants conjugated to DBCO-PEG20, DBCO-PEG30, and DBCO-HSA, respectively.

green fluorescent protein (sfGFP).1d The sfGFP is generally not immunogenic in an acute animal study, though it may be for a long period of time in vivo. Previously, we reported the site-specific conjugation of an albumin ligand fatty acid to sfGFP enhanced the serum half-life in mice, very likely via binding to mouse albumin in the blood.1d We expect that direct HSA conjugation to sfGFP will prolong the serum half-life in mice too. Furthermore, sfGFP level in the blood can be easily measured by standard enzyme-linked immunosorbent assay (ELISA) using anti-GFP antibody.1d

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MATERIALS AND METHODS

Materials. We purchased p-azido-L-phenylalanine (AzF) from Chem-Impex International (Wood Dale, IL). Ni-NTA agarose was obtained from Qiagen (Valencia, CA). Dibenzocyclooctyne-PEG4maleimide (DBCO-PEG4-MAL), dibenzocyclooctyne-mPEG molecules (20 kDa and 30 kDa), and dibenzocyclooctyne-PEG41812

DOI: 10.1021/acs.biomac.6b00238 Biomacromolecules 2016, 17, 1811−1817

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Biomacromolecules raphy−mass spectrometry (LC−MS) coupled with electron spray ionization (ESI). The injected samples were separated in an Accelar UHPLC system using an ACQUITY UPLC BEH C18 (1.7 μm) column at a flow rate of 400 μL/min with mobile phase consisting of water (0.1% formic acid) and acetonitrile (0.1% formic acid). The masses of peaks were analyzed by the LTQ-Orbitrap XL mass spectrometer (positive detection mode; spray voltage of 3.0 kV). The data were acquired by XCalibur (Thermo Scientific) and processed using ProMass deconvolution (Thermo Scientific). Site-Specific Conjugation of mPEG to sfGFP-AzF. The purified sfGFP-AzF (50 μM) was mixed with 100 μM of DBCO-mPEGs with 20 kDa and 30 kDa molecular weight (DBCO-PEG20 and DBCOPEG30) in PBS (pH 7.0) at 4 °C for 12 h to generate sfGFP-PEG20 or sfGFP-PEG30, respectively. After SPAAC reaction, the mixture was subjected to size exclusion chromatography by Ä KTAprime plus (GE Healthcare; Little Chalfont, Buckinghamshire, U.K.) using a Superdex 200 10/300 GL size exclusion column. Unreacted sfGFP-AzF was removed from elution fractions. Purified sfGFP-PEG20 and sfGFPPEG30 were concentrated by Vivaspin centrifugal filters (10 kDa molecular weight cutoff). Site-Specific Conjugation of Human Serum Albumin to sfGFP-AzF. High molecular aggregates of human serum albumin were removed by anion exchange chromatography using a HiTrap Q HP column. The purified HSA at 50 μM reacted with DBCO-PEG4-MAL at 200 μM in PBS buffer (pH 7.0) at room temperature for 2 h. To remove the unreacted linker, the reaction mixture was desalted with PBS buffer using a PD-10 column to obtain a HSA-linker conjugate, HSA-DBCO. The sfGFP-AzF (50 μM) reacted with HSA-DBCO at a 1:1 molar ratio in PBS buffer (pH 7.0) at room temperature for 7 h, and then subjected to a series of chromatographic separation. The sfGFP-HSA conjugate was isolated by cation exchange chromatography at pH 6.0 using a HiTrap SP HP column. The elute fractions containing the sfGFP-HSA conjugate were pooled and then desalted with PBS buffer (pH 7.0) using a PD-10 column. The eluent was concentrated using Vivaspin centrifugal filters (10 kDa molecular weight cutoff). The fluorescence of sfGFP samples (excitation, 480 nm; emission, 510 nm) was measured using a Synergy H1 four multimode microplate reader (BioTek, Winooski, VT). In Vivo Studies of Residual sfGFP in Serum. Mice were used in accordance with protocols approved by the Animal Care and Ethics Committees of Gwangju Institute of Science and Technology (GIST). The tail veins of young male BALB/c mice (n = 3) were intravenously injected with sfGFP-WT, sfGFP-HSA, sfGFP-PEG20, and sfGFPPEG30 (1.1 nmol in 100 μL PBS, pH 7.0). The blood was sampled at 0, 2, 4, 8, 12, 24, and 48 h post injection for sfGFP-WT and sfGFP conjugates via infraorbital blood collection. The sfGFP concentrations in the serum samples were measured by using a GFP-specific ELISA kit, according to the manufacturer’s protocol (Cell Biolabs, Inc., San Diego, CA).

transformed with two plasmids: pEVOL-pAzF and pQE80sfGFP_215amb. C321delA.exp E. coli host cells were genomically engineered to enhance amber suppression efficiency, resulting in a higher expression yield of a target protein. The E. coli transformants were incubated in 2xYT media supplemented with AzF to express sfGFP-AzF. The expressed sfGFP-AzF was purified according to Ni-NTA affinity chromatography, using a hexa-histidine tag attached to Cterminus of sfGFP-AzF. Wild-type sfGFP (sfGFP-WT) was also prepared as reported previously.1d The purified sfGFP-WT and sfGFP-AzF were analyzed by SDS-PAGE to check their purity and AzF incorporation. The both proteins exhibited the mainly single band at the expected site (Figure 2A, lanes 1 and 2 in

Figure 2. Validation of AzF incorporation in sfGFP-AzF. (A) Protein gel images of sfGFP and HSA species. sfGFP-WT (lane 1) and sfGFPAzF (lane 2) treated with a fluorescent dye, DBCO-PEG4carboxyrhodamine. HSA (lane 3) and DBCO-HSA (lane 4) treated with a fluorescent dye, coumarin-azide. The gel was exposed to UV (302 nm) to detect fluorescence (Fluorescence panel), and then stained with Coomassie blue (Coomassie Blue panel) to visualize proteins. (B) LC-MS spectra of sfGFP-WT (top) and sfGFP-AzF (bottom). a.u. indicates arbitrary unit.

Coomassie Blue panel) demonstrating a high purity. In order to confirm the reactivity of AzF inserted into sfGFP, both sfGFPWT and sfGFP-AzF were reacted with rhodamine-DBCO, a fluorescence dye containing a DBCO reactive functional group. After the reaction, both reaction mixtures were analyzed by SDS-PAGE. The fluorescence of protein bands was examined using a ChemiDoc XRS+ Bio-Rad imager. As expected, sfGFPAzF band exhibited fluorescence, while no fluorescence in sfGFP-WT band was detected (Figure 2A, lanes 1 and 2 in Fluorescence panel). Then, the identity of sfGFP-WT and sfGFP-AzF was further confirmed by LC-MS analysis. The deconvoluted LC-MS spectra of sfGFP-WT and sfGFP-AzF showed the peak at ca. 27,436 (m/z) and 27,624 (m/z), respectively (Figure 2B). As reported previously, methionine in the first position (Met1) of sfGFP is often cleaved in E. coli.1d It should be noted that the difference in masses between sfGFP-AzF and sfGFP-WT was 188 Da, which exactly matches with the mass of AzF, clearly indicating the insertion of AzF into sfGFP. The expected monoisotopic masses of Met1-cleaved sfGFP-WT and sfGFPAzF are 27,456 and 27 644 Da, respectively. The masses of sfGFP-WT and sfGFP-AzF experimentally determined were 0.07% off from the expected values. Such a small difference was likely due to nonoptimal mass calibration of the LC-MS equipment. Site-Specific Conjugation of HSA to sfGFP-AzF. The purified sfGFP-AzF was subjected to HSA conjugation. Since commercially available HSA has large aggregates, HSA was

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RESULTS AND DISCUSSION Site-Specific Incorporation of p-Azido-L-phenylalanine into sfGFP (Expression and Purification of sfGFPAzF). In order to compare the serum half-life extension of sfGFP upon HSA and PEG conjugation, we first prepared sfGFP-AzF, a sfGFP variant containing p-azido-L-phenylalanine (AzF). In order to exclude the effects of conjugation location/ number on the serum half-life extension, a single HSA or PEG molecule should be conjugated to the same site. As for sfGFP, we previously demonstrated that the insertion of p-ethynyl-Lphenylalnine (EthF) into the 215th position of sfGFP did not compromise the fluorescence, implying that the folded structure remained intact.1d Since both EthF and AzF are phenylalanine analogues containing a reactive functional group at para-position, we expected that insertion of AzF would not compromise the folded structure of sfGFP, leading to retained fluorescence. We achieved insertion of AzF into the 215th position of sfGFP using C321delA.exp E. coli host cells12 1813

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Figure 3. Conjugation of DBCO-HSA to sfGFP-AzF and purification of sfGFP-HSA conjugate. (A) Protein gel image of sfGFP-AzF (lane 1); sfGFP-AzF treated with DBCO-PEG4-carboxyrhodamine (lane 2); HSA (lane 3); DBCO-HSA treated with Coumarin azide (lane 4); and reaction mixture of sfGFP-AzF with DBCO-HSA (lane 5). The gel was stained with Coomassie blue to visualize proteins. (B) Cation exchange chromatogram of the reaction mixture of sfGFP-AzF and DBCO-HSA. The reaction mixture was loaded onto a SP-HP (5 mL) cation exchanger equilibrated with 20 mM Tris (pH 6.0) and eluted with NaCl gradient. (C) Fractions of sfGFP-HSA (F1) were isolated from the fractions of unreacted DBCO-HSA (FT) and sfGFP-AzF (F2) shown in the protein gel stained with Coomassie Blue.

purified by cation exchange chromatography as described in the Materials and Methods section. Then, a free sulfhydryl functional group in cysteine34 of the purified HSA reacted with a maleimide functional group of a heterobifunctional linker, DBCO-PEG4-MAL, to generate DBCO-HSA. The DBCO-PEG4-MAL linker has a clickable DBCO functional group at one end and a maleimide functional group at the other end, which was expected to cross-link sfGFP-AzF and HSA. In order to confirm the click chemistry reactivity of DBCO-HSA conjugate, the conjugate further reacted with Coumarin-azide. Both the purified HSA (66.5 kDa) and DBCO-HSA reacted with Coumarin-azide showed a band between bands of 50-kDa and 75-kDa molecular weight standards in the protein gel after Coomassie staining (Figure 2A, lanes 3 and 4 in Coomassie Blue panel). However, only the DBCO-HSA sample reacted with Coumarin-azide exhibited a band with fluorescence (Figure 2A, lanes 3 and 4 in Fluorescence panel), clearly indicating the click chemistry reactivity of DBCO-HSA toward azide functional group. The purified sfGFP-AzF and HSA-DBCO conjugate were coupled via strain-promoted azide−alkyne cycloaddition (SPAAC). After reaction, three bands were detected in the protein gel (Figure 3A, lane 5). The bottom two bands matched with those of the unreacted HSA-DBCO and unreacted sfGFP-AzF, respectively (Figure 3A). An additional band was detected between the bands of 100-kDa and 75-kDa molecular weight standards (Figure 3A, lane 5). Considering the molecular weight of sfGFP-AzF (27.6 kDa) and HSADBCO (66.5 kDa), the additional band was considered as a band of the sfGFP-HSA conjugate. In order to purify sfGFP-HSA, the reaction mixture of sfGFP-AzF and HSA-DBCO was subjected to cation exchange chromatography. The strong peak in the flow-through (FT) was expected to indicate the unreacted HSA-DBCO (Figure 3B and C). F1 fractions mainly contained sfGFP-HSA conjugate, while F2 fractions contained the unreacted sfGFP-AzF (Figure

3B and C). The F1 fractions were pooled to obtain a purified sfGFP-HSA, which was subjected to fluorescence measurement and animal studies. Site-Specific Conjugation of PEG Molecules to sfGFPAzF. For long circulation in blood, it has been revealed that the molecular weight and size of the polymer are important. In particular, it was previously reported that the kidney clearance limit of a linear polymer is around 40−45 kDa.15 Considering the molecular weight of sfGFP-AzF is around 27 kDa, we chose two methoxy PEG molecules with 20 and 30 kDa molecular weights as half-life extenders to achieve the significantly reduced kidney clearance. These PEG molecules, which have a DBCO functional group at one end, were denoted as DBCOPEG20 and DBCO-PEG30, respectively. These two PEG molecules were conjugated to sfGFP-AzF via SPAAC to generate sfGFP-PEG20 and sfGFP-PEG30 conjugates. Each reaction mixture exhibited an additional band above the band of sfGFP-AzF (Figure 4A, lanes 3−6). These additional bands were considered the bands of sfGFP-PEG20 and sfGFPPEG30. Apparent molecular weights of PEGylated sfGFPs were greater than expected values, because PEG molecules have greater hydrodynamic volume than proteins of a similar molecular weight.16 The conjugation yield of DBCO-PEG30 to sfGFP-AzF was lower than that of DBCO-PEG20 (Figure 4A), probably because a diffusion rate inversely correlates to a molecular weight of a macromolecule. The reaction mixtures were subjected to size exclusion chromatography to obtain pure sfGFP-PEG20 and sfGFP-PEG30. Upon PEG molecule conjugation of sfGFP-AzF, the molecular weight of sfGFPPEG conjugate increased by the molecular weight of the conjugated PEG molecule (ca. 20 or 30 kDa). Considering the molecular weight of sfGFP-AzF is approximately 28 kDa, the expected molecular weight of the sfGFP-PEG conjugate is ca. 48 or 58 kDa. Therefore, we applied size exclusion column chromatography to separate sfGFP-PEG conjugates from unreacted sfGFP-AzF. SDS-PAGE analysis of the fractions 1814

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the correctly folded structure, we measured the fluorescence of sfGFP-WT and sfGFP conjugates to evaluate whether conjugation perturbs the folded structure. The fluorescence (excitation, 480 nm; emission, 510 nm) of three conjugates and sfGFP-WT at the same concentration (ca. 4.0 μM) was measured in a 96-well plate using a fluorescence microplate reader. As shown in Figure 5B, all three conjugates (sfGFPPEG20, sfGFP-PEG30, and sfGFP-HSA) exhibited fluorescence comparable to that of sfGFP-WT. Even the fluorescence of sfGFP-HSA was about 10% higher than that of sfGFP-WT. These results clearly demonstrated that site-specific conjugation of an HSA/PEG molecule to sfGFP allows the retention or even enhancement of a critical property of sfGFP. Determination of the Serum Half-Lives of sfGFP Conjugates. Finally, the four sfGFP samples (sfGFP-WT, sfGFP-PEG20, sfGFP-PEG30, and sfGFP-HSA) were subjected to mice studies to determine the serum half-life. The sfGFP samples were intravenously administered into mice via tail vein. The residual serum concentrations of sfGFP-WT and sfGFP conjugates were measured by ELISA using an anti-GFP antibody recognizing free sfGFP-WT or the sfGFP portion in the sfGFP conjugates. The residual serum concentrations of sfGFP-WT and sfGFP conjugates vs time were plotted (Figure 6). The residual serum concentrations of sfGFP-WT were Figure 4. Conjugation of PEG molecules to sfGFP-AzF and purification of sfGFP-PEG conjugates. (A) Protein gel image of sfGFP-AzF (lane 1); sfGFP-AzF treated with DBCO-PEG4-carboxyrhodamine (lane 2); the reaction mixture of sfGFP-AzF with DBCOPEG20 (lanes 3 and 4) or DBCO-PEG30 (lanes 5 and 6). The gel was stained with Coomassie blue to visualize proteins. (B) Size exclusion chromatogram of the reaction mixture of sfGFP-AzF and DBCOPEG20. The reaction mixture was loaded onto a Superdex 200 10/300 GL size exclusion column equilibrated with PBS buffer (pH 7.0) and then eluted with PBS buffer. (C) Size exclusion chromatogram of the reaction mixture of sfGFP-AzF and DBCO-PEG30. Fractions of sfGFP-PEG20 or sfGFP-PEG30 (F1) were isolated from the fractions of unreacted sfGFP-AzF (F2) shown in the protein gel stained with Coomassie Blue.

Figure 6. Residual sfGFP in serum of intravenously injected sfGFPWT and three sfGFP conjugates (sfGFP-HSA, sfGFP-PEG20, and sfGFP-PEG30) in BALB/c mice (n = 3). The amounts of residual sfGFP samples were measured from blood samples drawn at different time points: 0, 2, 4, 8 h for sfGFP-WT; 0, 2, 4, 8, 12, 24, and 48 h for sfGFP conjugates. Residual sfGFP in the serum on a logarithmic scale over time was plotted to give a linear fit. Each data point represents the mean (n = 3) ± standard deviations. t1/2 indicates the serum half-life.

revealed that F1 fractions contained the sfGFP-PEG conjugates, while the F2 fractions mainly contained the unreacted sfGFPAzF (Figure 4B and C). The F1 fractions showing mainly sfGFP-PEG conjugates were pooled and further characterized. The four purified sfGFP variants (sfGFP-WT, sfGFP-HSA, sfGFP-PEG20, and sfGFP-PEG30) were analyzed by SDSPAGE (Figure 5A), indicating that these sfGFP variants/ conjugates were successfully purified to a high level of purity. We hypothesized that site-specific incorporation of an HSA or PEG molecule to a permissive site of sfGFP will not perturb the folded structure of sfGFP. Since fluorescence of sfGFP requires

negligible 8 h after injection. The serum half-life of sGFP-WT was 1.5 h (Figure 6), which is comparable to the half-life previously reported.1d The serum half-lives of sfGFP-PEG20, sfGFP-PEG30, and sfGFP-HSA were 11.0, 14.3, and 13.5 h,

Figure 5. Protein gel image and relative fluorescence of four purified sfGFP samples (sfGFP-WT, sfGFP-HSA, sfGFP-PEG20, sfGFP-PEG30). (A) sfGFP-WT (lane 1), sfGFP-PEG20 (lane 2), sfGFP-PEG30 (lane 3), and sfGFP-HSA (lane 4) were analyzed by SDS-PAGE, and stained with Coomassie staining. (B) Fluorescence of the sfGFP samples relative to that of sfGFP-WT. 1815

DOI: 10.1021/acs.biomac.6b00238 Biomacromolecules 2016, 17, 1811−1817

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of the serum half-life with additional benefits of low immune response and good degradability. Handling or chemical modification of HSA during the albumination process may lead to slight denaturation, which may be sufficient to generate an immune response. Therefore, more studies on immune responses of denatured HSA would be required to further develop albumination as a tool to enhance the serum half-life.

respectively (Figure 6). Compared to the serum half-life of sfGFP-WT (1.5 h), the serum half-lives of sfGFP-PEG20, sfGFP-PEG30, and sfGFP-HSA were extended 7.3, 9.5, and 9.0 times, respectively, clearly demonstrating that conjugation of HSA or PEG molecules significantly enhances the serum halflife of a target protein. Considering the standard deviation of these serum half-lives, the serum half-life of sfGFP-HSA was comparable to those of sfGFP-PEG conjugates. It is noteworthy that the serum half-life extension of HSA conjugate is expected to be much greater in humans compared to mice. It was already reported that the binding affinity of HSA to a mouse FcRn is several times weaker than to a human FcRn.17 Therefore, assuming the evasion of intracellular degradation of HSA (or HSA-conjugated proteins) is the main mechanism for the prolonged serum half-life in humans, it is likely that the serum half-life of a HSA-conjugated protein is much greater than that of a PEG-conjugated protein in humans. It was previously reported that albumination of therapeutic peptides/proteins (glucagon-like peptide-1, granulocyte-colony stimulating factor, and interferon alpha-2b) extended the serum half-life compared to unmodified ones in humans 6 to 10 times more than in mice.5c However, in order to demonstrate this, more extensive pharmacokinetic studies in humans are required in the future. In order to apply HSA-conjugation to other therapeutic proteins, we need to consider a significant volume and rigid shape of HSA in contrast to a flexible shape of PEG. Therefore, depending on a conjugation site on a target therapeutic protein, a conjugation yield will vary significantly and the HSAconjugation may perturb the structure and therapeutic activity of a target protein. In order to circumvent these issues, sitespecific conjugation of a HSA molecule to a permissive site of a therapeutic protein can be used. We previously demonstrated that site-specific conjugation technique led to albumination of urate oxidase with four subunits (total 135 kDa) without losing its therapeutic activity.1c

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AUTHOR INFORMATION

Corresponding Author

*Phone: +82 62-715-2312. Fax: +82 62-715-2304. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge financial support from the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (Grant No. 2014R1A2A1A11050322). This research was partially supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014H1C1A1067014). The authors are grateful to Eun Sil Choi for help on chromatographic purification of proteins.

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REFERENCES

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CONCLUSIONS As a half-life extender, HSA has several beneficial features, such as very low immune response and good degradability. In this study, we investigated whether HSA is an effective serum halflife extender as an alternative to popular PEG molecules. Sitespecific incorporation of AzF into sfGFP and bioorthogonal click chemistry achieved the site-specific conjugation of HSA or PEG molecules to sfGFP, generating three novel sfGFP conjugates (sfGFP-HSA, sfGFP-PEG20, and sfGFP-PEG30). Standard protein purification chromatography was applied to purify sfGFP conjugates in a high purity. The three sfGFP conjugates (sfGFP-HSA, sfGFP-PEG20, and sfGFP-PEG30) exhibited the fluorescence comparable to or even greater than that of sfGFP-WT. Therefore, we concluded that HSA or PEG molecules can be conjugated to a target protein without compromising the critical property of the target protein. In mice, the serum half-lives of sfGFP-HSA, sfGFP-PEG20, and sfGFP-PEG30 were 13.5, 11.0, and 14.3 h, which were 9.0, 7.3, and 9.5 times greater than that of sfGFP-WT, respectively. These results indicated that the extension of the serum half-life of sfGFP upon HSA conjugation is comparable to those of relatively large PEG molecules (20 kDa and 30 kDa). Combined with the previous case of uricase,1c these results also indicated that HSA conjugation seems a general approach to enhance the serum half-life of a protein in vivo. Finally, we concluded that HSA can be an alternative to PEG for extension 1816

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Article

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DOI: 10.1021/acs.biomac.6b00238 Biomacromolecules 2016, 17, 1811−1817