Sortase-catalyzed transformations thatimprove the properties of cytokinesMaximilian W. Poppa,b, Stephanie K. Dougana, Tzu-Ying Chuanga, Eric Spoonera, and Hidde L. Ploegha,b,1

aWhitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142; and bDepartment of Biology, Massachusetts Institute ofTechnology, 77 Massachusetts Avenue, Cambridge, MA 02142

Edited by Donald F. Steiner, University of Chicago, Chicago, IL, and approved January 3, 2011 (received for review November 9, 2010)

Recombinant protein therapeutics often suffer from short circulat-ing half-life and poor stability, necessitating multiple injectionsand resulting in limited shelf-life. Conjugation to polyethylene gly-col chains (PEG) extends the circulatory half-life of many proteins,but the methods for attachment often lack specificity, resultingin loss of biological activity. Using four-helix bundle cytokines asan example, we present a general platform that uses sortase-mediated transpeptidation to facilitate site-specific attachmentof PEG to extend cytokine half-life with full retention of biologicalactivity. Covalently joining the N and C termini of proteins to obtaincircular polypeptides, again executed using sortase, increases ther-mal stability. We combined both PEGylation and circularizationby exploiting two distinct sortase enzymes and the use of a mole-cular suture that allows both site-specific PEGylation and covalentclosure. The method developed is general, uses a set of easilyaccessible reagents, and should be applicable to a wide varietyof proteins, provided that their termini are not involved in receptorbinding or function.

Many clinically relevant cytokines share a four-helix bundlestructure, typified by IFNα2, Granulocyte colony-stimulat-

ing factor 3 (GCSF-3), Erythropoietin (EPO), IL-2, IL-4, IL-7,IL-9, and IL-15. Cocrystal structures of cytokines withreceptor fragments and biochemical studies that map residuescritical for interaction of a cytokine with its receptor show thatthe receptor contacts the sides of the helical bundles (1). Thismode of interaction positions the N and C termini of the cytokineaway from the receptor.

Polyethylene glycol chains attached to therapeutically impor-tant proteins increase circulatory half-life, reduce clearance bykidney filtration, reduce proteolysis, and reduce the generationof neutralizing antibodies (2–4). The attachment of PEG com-monly employs standard chemistries that target reactive aminoacid side chains (e.g., cysteine and lysine). This strategy often gen-erates a heterogeneous mixture in which multiple amino acids inthe target are modified with a PEG chain, necessitating cumber-some separations and characterization (5). Such PEGylatedmolecules often show decreased biological activity, likely due toattachment of a PEG chain to a residue important for interactionwith the receptor (6)-this problem may be overcome by site-specific PEGylation. Although engineering of a carefully placedunpaired cysteine residue allows site-specific PEGylation (7, 8),this method must be tailored to the specific protein target.

Sortase A from Staphylcoccus aureus (SrtAStaph) is a thiol-containing transpeptidase that recognizes an LPXTG motif inmultiple structurally unrelated substrates (9). SrtAStaph cleavesthe peptide bond between the threonine and glycine residues withconcomitant formation of a thioacyl enzyme intermediate thatinvolves the catalytic cysteine and the substrate threonine. Thisacyl-enzyme is resolved by nucleophilic attack by the N terminusof an oligoglycine peptide, resulting in formation of an amidebond between the substrate protein and the incoming nucleophile(10). Sortase A tolerates C-terminal extensions of the oligogly-cine nucleophile, allowing diverse functionalized nucleophilesto be installed site specifically onto proteins equipped with anLPXTG motif (11, 12). The related Streptococcus pyogenes

sortase accepts di-alanine based nucleophiles, which the S. aureusenzyme does not. This sortase (SrtAstrep) cleaves the LPXTAmo-tif between theronine and alanine and allows installation of mod-ified alanine-based nucleophiles. SrtAstrep also recognizes andcleaves LPXTG motifs, albeit with reduced efficiency, howeverthe LPXTA motif is refractory to cleavage by SrtAStaph (13, 14).

Here we present a general strategy for site-specific modifica-tion of therapeutic recombinant proteins at their termini, anapproach that is particularly well suited to the four-helix bundlecytokines, where the termini are distant from the receptor inter-action site. We show improvements in thermal stability by cova-

Fig. 1. The sortase reaction scheme. Cyclization of substrates equippedwith a C-terminal LPXTG sortase recognition element as well as an N-terminalglycine by SrtAStaph (left). Substrates lacking the N-terminal glycine andincubated in the presence of exogenous oligo-glycine functionalized PEGare site-specifically PEGylated (right). Circular, PEGylated proteins can beconstructed by incubating substrates bearing an N-terminal glycine and aC-terminal SrtAstrep cleavage site (LPXTA) with an alanine-based nucleophilecarrying an amino oxy group and the SrtAStaph cleavage site (Center). Thistranspeptidation product is cyclized upon incubation with SrtAStaph. Finally,PEG is attached by aniline-catalyzed oxime ligation with methoxy-cappedPEG propionaldehyde. The oxime bond formed is shown (inset).

Author contributions: M.W.P. and H.L.P. designed research; M.W.P., S.K.D., T.-Y.C., andE.S. performed research; M.W.P., S.K.D., and E.S. analyzed data; and M.W.P. and H.L.P.wrote the paper.

Conflict of interest statement: The authors have applied for a patent based on the tech-nology used in the paper.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: ploegh@wi.mit.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1016863108/-/DCSupplemental.

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lently joining the N and C termini of these proteins (15). Wepresent a general method for combining both site-specificPEGylation and improved thermal stability in a single moleculeby using a molecular suture produced in two successive rounds ofsortase-catalyzed transpeptidation (Fig. 1).

ResultsModification of IFNα2. We first applied the sortase reaction tohuman interferon alpha (IFNα2), a four-helix bundle cytokinewhere chemical PEGylation results in multiple positional isomersand a ∼90% decrease in biological potency (16). We expressedin Escherichia coli two versions of IFNα2, both with an LPETGsortase motif immediately followed by a hexahistidine tag for easeof purification. At the amino terminus, one of the two proteinspossesses a diglycine motif for cyclization. This material caneither be cyclized or PEGylated at the C terminus (Fig. 2A).IFNα2 with an N-terminal diglycine motif and a C-terminalLPETG was successfully cyclized by incubation with SrtAStaphand purified to homogeneity by ion-exchange chromatography.The mass of the circularized product in a crude sortase reactiondiffers from a linear hydrolysis product by −18 Da, consistentwith an intramolecular transpeptidation reaction (Fig. 2B). Weidentified the unique junction peptide that arises from intra-molecular cyclization by tandem mass spectrometry (MS/MS)analysis of tryptic digests (Fig. 2C). Site-specific PEGylation ofthe IFNα2 variant that lacks the N-terminal diglycine motif (and

thus unable to undergo intramolecular cyclization) was achievedby incubation with sortase and a GGGK peptide to which a10 kDa PEG moiety was affixed via the lysine ϵ-amine (Probe 1,Fig. 2A and Fig. S1A). We confirmed equipotent biological activ-ity of all four interferon alpha variants in a Daudi cell prolifera-tion inhibition assay when compared to a commercially available,non-PEGylated preparation (Fig. 2D and Table S1). Addition ofa protein-sized PEG module to the C terminus of IFNα2 doesnot appreciably perturb receptor binding and biological potency.We hypothesized that the circular IFNα2 would be more resistantto thermal denaturation, because the termini are clamped shutand thus should not “fray” (15, 17). We measured thermal dena-turation with a Thermofluor assay, using the commerciallyavailable Sypro Orange dye (18). Indeed, the circular form ofIFNα2 had a significantly elevated melting temperature (Tm)compared to the other three variants, all of which exhibitednearly identical Tm’s (Fig. 2E, Table S1, and Fig. S2A). Finally,we injected mice with either the linear IFNα2 variant used to con-struct the PEGylated form, or the IFNα2 variant bearing a 10 kDaPEG chain (Probe 1) at its C terminus and measured the serumlevels over time by ELISA. The PEGylated form decayed withslower kinetics than its non-PEGylated counterpart (Fig. 2Fand Table S1).

Modification of a Single Protein Preparation by Multiple Nucleophiles.The sortase platform offers the ability to install nearly any

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Fig. 2. Synthesis and characterization of humanIFNα2 conjugates. (A) GG-IFNα-LPETGGHis6 was usedto obtain backbone-cyclized IFNα2, and IFNα-LEPTGGHis6 was used to make IFNα-PEG (10 kDa),bearing a C-terminal PEG chain. Purified proteinswere resolved by 12.5% SDS-PAGE and stained withCoomassie. (B) ESI-MS characterization of IFNαspecies in crude sortase reactions either before (linearGG-IFNα-LPETGGHis6) or after (cylic IFNα2) overnightincubation at 25 °C. (C) MS/MS identification of atryptic peptide comprising the IFNα2 C terminus,followed by the SrtAStaph cleavage site, joined tothe N-terminal glycines in the IFNα2 precursor. (D) Invitro bioactivity of IFNα conjugates in a Daudi cellproliferation inhibition assay. Dose-response curvesfor Daudi cell proliferation inhibition were measuredby [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium] bromide (MTT) assay, in triplicate, with thestandard deviation displayed. (E) Resistance of back-bone-cyclized IFNα to thermal denaturation. Changein Tm for circular and PEGylated IFNα conjugates,relative to their precursor proteins, were measured bythermal shift assay, with n ¼ 4. (F) Change in serumIFNα levels following tail vein injection of site-specifically PEGylated IFNα as well as its precursorprotein. 10 μg of each protein was injected and, atthe indicated time points, blood was withdrawnand cytokine concentration in serum was determinedby ELISA assay. Data are means � standard deviationfor three mice in each group.

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nongenetically encoded entity onto the C terminus of a singlepreparation of recombinant protein. We demonstrate this abilityby synthesizing a nucleophile that bears a 20 kDa PEG moiety(Probe 2 and Fig. S1A), as well as the previously described10 kDa PEG probe (Probe 1), and appended them to the iden-tical preparation of IFNα2 (Fig. 3A). Both conjugates were testedfor biological activity (Fig. 3B) and are highly potent (Table S2).

Superior Properties Endowed upon Sortase Substrates Are Applicableto Multiple Proteins. Sortase allows the installation of the samenonnatural group onto different proteins, unrelated in sequenceor amino acid composition. Accordingly, we applied the sortasereaction to a different four-helix bundle cytokine, GCSF-3, withsimilar results. We first made the linear precursor GCSF variantsand then cyclized or PEGylated them with probe 1 using sortase(Fig. 4A). All variants possess equal or superior in vitro bioactiv-ity relative to a commercial non-PEGylated preparation, asassessed by a standard cell proliferation assay with the NFS-60murine myeoblastic cell line (19) (Fig. 4B and Table S3). Likecyclic IFNα2, cyclic GCSF-3 shows increased thermostability(Fig. 4C, Fig. S2B, and Table S3) and PEGylated GCSF-3 hasa significantly extended circulatory half-life relative to its non-PEGylated precursor (Fig. 4D and Table S3) upon injection intomice. PEGylated GCSF-3 also led to a more robust and pro-longed proliferation of granulocytes in injected mice than itsnon-PEGylated analog (Fig. 4E).

Modification of Glycoproteins. Many important therapeutics areglycosylated proteins that traverse the secretory pathway and areproduced in mammalian cell culture. Are such proteins equallyamenable to engineering using a sortase-mediated transpeptida-tion reaction? We chose human erythropoietin (EPO) because

∼40% of its mass is composed of bulky, charged N-linked glycans,and cyclization provides a stringent test of the sortase method, asit requires (minimal) modifications at both termini. We replacedthe endogenous EPO signal sequence with the murine H-2Kb

signal sequence, followed by two glycine residues. This signalpeptide is cleaved in mammalian cells with concomitant exposureof an N-terminal glycine residue. We added the requisite sortasemotif followed by a hexahistitdine tag at the C terminus of EPOfor ease of purification. Human embryonic kidney (HEK)-293Tcell lines stably transduced with this construct yielded prepara-tions that were <50% pure after a single round of Nickel-nitrilo-triacetic acid immobiliized metal affinity chromatography (Ni-NTA IMAC) of conditioned medium. Material from such pre-parations can be cyclized successfully upon incubation withsortase (Fig. 5A). Again, we identified the unique junction pep-tide that arises from covalent ligation of the N and C termini byMS/MS analysis of cyclized EPO, shorn of all glycans by incuba-tion with peptide: N-glycosidase F (PNGaseF), and digestedwith trypsin (Fig. 5B). Both the linear preparations as well asthe sortase reaction containing cyclic EPO were tested in a cellproliferation assay with Ba/F3 cells that stably express the ery-thropoietin receptor (20) and these compared favorably withcommercial EPO preparations. Most importantly, the cyclic formis as potent as its linear counterpart (Fig. 5C and Table S4),indicating that modification of the EPO termini does not affectits biological activity.

Two-Step Transacylation Allows Combination of Enhanced Properties.Having shown for multiple examples that sortase-mediatedsite-specific modification of the C terminus with PEG increasescirculatory half-life with nearly no loss of biological activity,and that covalent closure of the N and C termini yield proteinsstabilized against thermal denaturation, we executed a schemethat combines these desirable properties (Fig. 1). In the first step,we exploit a previously described sortase from Streptococcus pyo-genes (13, 14) that accepts alanine-based nucleophiles to affix apeptide containing a nonnatural amino oxy containing aminoacid, followed by the SrtAStaph cleavage site. We use SrtAStaph toeffectuate covalent closure, as the LPETAA site left by SrtAstrep isresistant to attack by SrtAStaph, to yield a circular protein with theamino oxy group at the place of suture. Next, the amino oxy groupis used in a bioorthognal oxime ligation reaction (21–23) withcommercially available methoxy-capped PEG-propionaldehyde(Fig. S1B). We recombinantly expressed IFNα2 bearing twoglycines at the N terminus, and at the C terminus the SrtAstrepcleavage site, followed by a hexahistidine tag. The purifiedprotein was subjected to the two-step transacylation procedure toyield circular IFNα2 bearing the amino oxy group. After ionexchange chromatography, this material was PEGylated and pur-ified by cation exchange chromatography (Fig. 6A). Withoutoptimization, the final yield of circular PEGylated protein was∼10%, because of the multiple separate chromatography and pro-tein concentration steps involved in production and PEGylation.The mass of the material before oxime ligation was consistentwith its cyclization (Fig. S3A). Digestion with AspN and MS/MS analysis revealed the unique peptide that arises as a conse-quence of backbone cyclization consisting of the molecular sutureprobe stitched between the C and N termini of IFNα (Fig. 6B).All three variants yielded virtually indistinguishable IC50 values inan inhibition of cell proliferation assay (Fig. 6C and Table S5) andthe circular versions were stabilized against thermal denaturation(Fig. 6D, Table S5, and Fig. S2C). The circular versions remainedbiologically potent after boiling when given the opportunity torefold, whereas we were unable to extract an IC50 value for theboiled linear preparation over the concentration range assayed(Fig. 6E and Table S6). We injected the linear and the circular,PEGylated forms of IFNα2 into the tail vein of mice and mea-sured half life by ELISA. The circular, PEGylated species was

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Fig. 3. Sortase installs multiple probes onto a single preparation of protein.(A) The same preparation of IFNα-LEPTGGHis6 was PEGylated by sortase re-action with either a 10 kDa or a 20 kDa PEG nucleophile. Purified proteinswere resolved by 12.5% SDS-PAGE and stained with Coomassie. (B) In vitrobioactivity of 10 kDa and 20 kDa PEGylated IFNα conjugates in a Daudi cellproliferation inhibition assay. Dose-response curves for Daudi cell prolifera-tion inhibition were measured by MTT assay, in triplicate, with the standarddeviation displayed.

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cleared significantly more slowly than the linear form (Fig. 6F andTable S5).

DiscussionThe sortase transpeptidase reaction allows facile site-specificPEGylation of multiple distinct proteins. In all cases tested, thesite-specific C-terminal PEGylation proceeds efficiently (Fig. S3B)and yields adducts of known stoichiometry that are biologicallyequipotent to the non-PEGylated versions, but retain the in-crease in circulatory half life associated with PEG modification.We attribute these properties to the fact that all enzymatic trans-formations are performed on native proteins, procedures thatshould not affect overall conformation or exposure of function-ally important side chains. Because our approach to PEGylationis site specific, all preparations are also homogenous and easilypurified (5). Although we limit our examples to four-helix bundlecytokines, this platform should be readily extended to structurallydistinct therapeutic proteins, with the singular requirement thatthe C terminus is not involved in receptor binding. A sortase-based approach requires the genetic fusion of a very small (fiveamino acid) tag to the protein of interest and all transformationsoccur under native conditions. The additional amino acids thatresult from fusion to the sortase recognition motif are close tothe site of PEG attachment. Immunogenicity of this site is verylikely reduced by the PEG moiety shielding this area (3). We andothers have labeled proteins with an exposed N-terminal glycineusing sortase (14, 24). This approach should therefore be readilyextended to site-specific PEGylation at the N terminus as well.

Cyclization of the backbone of protein-based therapeuticsalso proceeds efficiently (Fig. S3B) and yields preparations thatare more resistant to thermal denaturation. Cyclic proteins arealso resistant to exoproteolytic attack (25, 26), a feature thatmay enhance utility of any therapeutic proteins exposed to exo-proteases, for example upon receptor mediated internalization.In addition, cyclization of proteins and peptides has been shownto improve potency, stability, and oral bioavailability (27–30).

Finally, we have inserted a nontemplate encoded entity inwhat is topologically internal to a circular protein, a feat thatcannot be accomplished genetically by intein-based methods orby any other currently known means. More generally, this dualtransacylation scheme can be used to insert nonnatural groupsbetween fully native proteins expressed separately, or betweenprotein domains. We suspect that this approach will find applica-tion in the protein engineering field and so further extend theutility of sortases as tools.

Experimental ProceduresSortase Reactions. Sortase reactions with SrtAStaph were perform-ed as described previously (11). For either cyclization or PEGyla-tion, reactions containing 50 μM substrate, 50 μM SrtAStaph and1 mM probe (for PEGylation) were incubated overnight at 25 °Cwithout agitation. Reactions were purified by cation exchangechromatography on a Mono-S (GE Healthcare). For IFNa2reactions, continuous gradient chromatography was performedwith 20 mM 2-(N-morpholino) ethane sulfonic acid (MES) pH5.0 and 20 mM MES, 1 M NaCl pH5.0, as eluent. For GCSF re-actions, continuous gradient chromatography was performed

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EFig. 4. Synthesis and characterization of humanGCSF-3 conjugates. (A) GG-GCSF-LPETGGHis6 was usedto make backbone cyclized GCSF-3, and GCSF-LEPTGGHis6 was used to make GCSF-PEG (10 kDa),bearing a C-terminal PEG chain. Purified proteinswere resolved by 12.5% SDS-PAGE and stained withCoomassie. (B) In vitro bioactivity of GCSF-3 conju-gates in an NFS-60 cell proliferation assay. Dose-response curves for NFS-60 cell proliferation weremeasured by MTT assay, in triplicate, with the stan-dard deviation displayed. (C) Resistance of backbonecyclized GCSF-3 to thermal denaturation. Change inTm for circular and PEGylated GCSF-3 conjugates, re-lative to their precursor proteins, were measured bythermal shift assay (n ¼ 4). (D) Change in serum GCSFlevels following tail vein injection of site-specificallyPEGylated GCSF-3, as well as its precursor. 5 μg of eachprotein was injected and, at the indicated time points,blood was withdrawn and cytokine concentration inserumwas determined by ELISA assay. Data aremeansfrom three mice in each group, with the standarddeviation displayed. (E) GCSF, PEGylated via sortase,is highly potent in vivo. C57BL/6 mice (n ¼ 4 pergroup) were injected intravenously with linear GCSF,GCSF-PEG (10 kD), or saline control (PBS). Peripheralbloodwas collected at the indicated times and stainedwith antibodies to Gr-1 and CD11b. Newly generatedgranulocytes were defined as Gr-1 intermediateCD11b+ cells (31).

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with 20 mM sodium acetate pH 4.5 and 20 mM sodium acetate,1 M NaCl pH 4.5, as eluent. Peaks containing the desired productwere pooled, concentrated with Vivaspin 500 centrifugal concen-trators (Sigma) and protein concentration was determined byBradford Assay (BioRad). For EPO cyclization, 300 μL ofNi-NTA elutate (0.101 mg∕mL total protein) was incubated with100 μM SrtA and sortase buffer in 400 μL total volume at 25 °Cfor 16 h. For two-step transacylation reactions, 150 μM substratewas incubated with 50 μM SrtAstrep and 2 mM probe in 100 mMTris, 150 mM NaCl pH 8.0 for 4 h at 37 °C. SrtAstrep activitywas halted by incubation with 500 μM of the protease inhibitorE-64 on ice for 1 h. Crude reaction mixtures were then supple-mented with 10 mM imidazole and subjected to Ni-NTA chroma-tography (Qiagen) to remove prematurely cyclized material.Protein was eluted with 50 mM Tris, 150 mM NaCl, 500 mMimidazole, and buffer exchanged into SrtAStaph buffer (50 mMTris, 150 mM NaCl, and 10 mM CaCl2) with a PD10 desaltingcolumn (GE Healthcare). For cyclization, this material wasincubated overnight with 50 μM SrtAStaph and purified by cationexchange as described for other IFNα2a reactions. Concentrated,purified circular IFNα2 bearing theAminooxyacetic acid (AOAA)group was then diluted with 1 volume of 50 mM sodium acetatepH 4.5, 150 mM NaCl containing 2 mM methoxy-capped PEG-

propionaldehyde, and 100 mM aniline (JT Baker) and incubatedat 30 °C for 3 h without agitation. This reaction mixture wasagain purified by cation exchange chromatography, concentrated,and protein concentration was determined by Bradford assay.

Thermal Denaturation Assays. Thermal shift assays were performedin a Roche 480 lightcycler and fluorescence was measured withExcitation ¼ 533 nm, Emission ¼ 610 nm. For GCSF-3, 1 μg ofeach variant was mixed with 1 μL of 100× Sypro orange in 25 μLtotal volume of 50 mM acetate pH 4.0, 150 mM NaCl. n ¼ 4 forall samples with buffer as a blank control and lysozyme as apositive control. For IFNα2, 1 μg of each variant was mixed with

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Fig. 5. Synthesis and characterization of cyclic human erythropoietin.(A) GG-EPO-LPETGGHis6 bearing the H-2Kb signal sequence was expressedin HEK-293T cells. Conditioned media was subjected to Ni-NTA IMAC purifi-cation. Eluted material was supplemented with 100 μM SrtAStaph and incu-bated at 25 °C. Aliquots were removed at the indicated time points,subjected to 12.5% SDS-PAGE and protein was visualized by silver staining.(B) MS/MS identification of a peptide generated by tryptic digestion contain-ing the C terminus of EPO, followed by the SrtAStaph cleavage site andjoined to the N terminus of EPO. (C) In vitro bioactivity of linear and cylicEPO conjugates in a cell proliferation assay using BaF3 cells stably expressingthe erythropoietin receptor (BaF3-EPOR). Eluted material from Ni-NTA IMACpurification was incubated in the presence or absence of sortase for 16 h at25 °C and these crude reactions were measured for in vitro EPO bioactivity.Dose response curves for BaF3-EPOR cell proliferation were measured byMTTassay, in triplicate, with the standard deviation displayed. Concentrationswere not adjusted for impurities in the EPO preperations.

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Fig. 6. Synthesis and characterization of cyclic, PEGylated IFNα2. (A) GG-IFNα-LPETAAHis6 was used to make backbone-cyclized IFNα2 with the aminooxy moiety stitched between the site of closure. This material was then usedto generate cyclic, PEGylated IFNα2 by aniline catalyzed oxime ligation. Pur-ified proteins were resolved by 12.5% SDS-PAGE and stained with Coomassie.(B) MS/MS identification of a peptide generated by AspN digestion contain-ing the C terminus of IFNα, followed by the alanine-based probe joined to theN terminus of IFNα by SrtAStaph. Note that the amino oxy group was likely lostduring MS/MS analysis and ESI-MS reconstructions of unfragmented proteinrevealed a mass consistent with installation of the amino oxy group (Fig. S3).(C) In vitro bioactivity of linear and cyclic IFNα conjugates in a Daudi cell pro-liferation inhibition assay. Dose-response curves for Daudi cell proliferationinhibition were measured by MTT assay, in triplicate, with the standard de-viation displayed. (D) Resistance of circular-AOAA and circular-PEGylatedIFNα to thermal denaturation. Change in Tm’s for circular-AOAA and circular-PEGylated IFNα conjugates, relative to their precursor protein, were mea-sured by thermal shift assay (n ¼ 4). (E) Circular cytokines remain biologicallyactive after boiling. The indicated variants were diluted to 1 mg∕mL inRPMI∕10% serum, boiled for 4 min, allowed to cool at room temperaturefor 16 h, and assayed for inhibition of Daudi cell proliferation. (F) Changein serum IFNα levels following tail vein injection of circular, site-specificallyPEGylated IFNα as well as its linear precursor protein. 10 μg of each proteinwas injected and, at the indicated time points, blood was withdrawn andcytokine concentration in serum was determined by ELISA assay. Data aremeans from three mice in each group, with the standard deviation displayed.

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1 μL of 100× Sypro orange (Invitrogen) in 25 μL total volumeof 50 mM MES ph 5.0, 150 mM NaCl. n ¼ 4 for all sampleswith buffer as a blank control and lysozyme as a positive control.Samples were heated from ambient temperature to 95 °C at0.01 °C∕s with 6 acquisitions∕°C. The included Roche softwarewas used to calculate the negative first derivative of the tempera-ture change as a function of time and the minima were used as theTm. Because of the impurities in the cyclic EPO preperations, Tmwas not determined.

Cell-Based Bioactivity Assays. Daudi cell proliferation inhibitionassays to measure the activity of IFNα2 conjugates were per-formed as described (8) and compared to commercial IFNα2(PBL Interferon Source) with low passage Daudi cells (ATCC),cultured in RPMI medium 1640∕10%Heat inactivated fetalcalf serum ðIFSÞ∕50 units∕mL penicillin, 50 μg∕mL streptomy-cin sulfate. Cell proliferation was measured by MTTassay accord-ing to manufacturer’s directions (ATCC). For the denaturationassay, samples diluted to 1 μg∕mL in RPMI medium 1640∕10%IFS were boiled for 4 min, allowed to cool at room temperaturefor 16 h, and assayed for Daudi cell proliferation inhibition.NFS-60 cell proliferation assays to measure G-CSF conjugateactivity were performed as follows. NFS-60 cells (a kind gift fromJames Ihle, St. Jude Children’s Research Hospital) were culturedin RPMI medium 1640∕10%IFS∕50 units∕mL penicillin, 50 μg∕mL streptomycin sulfate supplemented with murine IL-3(20 U∕mL, R&D Systems). Cells were washed extensively incomplete RPMI medium 1640 medium lacking IL-3 and resus-pended at 1 × 105 cells∕mL in complete RPMI medium 1640lacking IL-3. Titrated GCSF conjugates (50 μL) in completeRPMI medium 1640 were aliquoted into a flat bottom 96 wellplate and 50 μL of cells (0.5 × 104 cells∕well) were added to eachwell. Cells were cultured for 3 d and an MTTassay (ATCC) wasperformed according to manufacturer’s directions. Each platecontained a titration of commercial GCSF3 (Peprotech) andreadings were blanked against wells containing only NFS-60cells. The activity of each conjugate was measured a minimumof n ¼ 3 times. BaF3 cells stably expressing the EPO receptor(BaF3 EPO-R cells, a kind gift from Harvey Lodish, Whitehead

Institute) were used to measure EPO activity. BaF3 EPO-R cellswere cultured as described for NFS-60 cells and assays wereperformed essentially as described for G-CSF, except EPO circu-larization reactions incubated with or without sortase for 16 hat 37 °C were used. The total protein concentration of the EPOinput material (measured by the Bradford method) was used andwas not adjusted for impurities. Commercial preparations ofEPO (eBioscience) were used as standards and readings wereblanked against BaF3 EPO-R cells cultured with no cytokines.Activities were measured a minimum of n ¼ 3 times.

Circulatory Half-Life Assays. For circulatory half-life assays, micewere injected in the tail vein with each protein (10 μg per mousefor IFNα2 conjugates, 5 μg per mouse for G-CSF conjugates)and subjected to retroorbital eye bleed at the indicated timepoints. Blood was harvested, centrifuged at 1;957xg in a tabletopcentrifuge, and serum was collected and snap-frozen. Elisa assaysto measure the quantity of cytokine in serum samples wereperformed according to the manufacturer’s directions (IFNαfrom PBL Interferon Source, G-CSF from Invitrogen). Eachconjugate was injected into n ¼ 3 mice. Data was fit to a twophase exponential decay in GraphPad Prism.

Granulocyte Proliferation Assay. Peripheral blood was collectedretroorbitally into EDTA collection tubes. Red blood cells werelysed in hypotonic lysis buffer and the remaining peripheralblood mononuclear cells were stained with anti-myeloid differen-tiation antigen-1 (Gr-1) antibody labeled with phycoerythrin(PE) (BD Pharmingen), anti-cluster of differentiation 11bprotein antibody labeled with FITC (anti-CD11b-FITC) (BDPharmingen), and 7-Aminoactinomycin D (7-AAD) (ViaProbe,BD Pharmingen). Cells were analyzed using a FACS Caliber flowcytometer (BD).

ACKNOWLEDGMENTS.We thank Drs. Nick Yoder, John Antos, and Annemarthevan der Veen, for helpful discussions, and Drs. Harvey Lodish and James Ihlefor reagents. This work was supported by grants from the National Institutesof Health (NIH).

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