Enzyme-Mediated Site-Specific Antibody−Protein Modification Using a

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Enzyme-Mediated Site-Specific Antibody-Protein Modification Using a ZZ Domain as a Linker Takayuki Sakamoto,† Shiori Sawamoto,† Tsutomu Tanaka,*,‡ Hideki Fukuda,‡ and Akihiko Kondo† Department of Chemical Science and Engineering, Graduate School of Engineering, and Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan. Received April 28, 2010; Revised Manuscript Received October 18, 2010

A ZZ domain (ZZ) and alkaline phosphatase (AP), luciferase (Luc), or glucose oxidase (GOD) were conjugated using Sortase A (SrtA) from Staphylococcus aureus. The specific peptidyl linker for SrtA was genetically fused to the C-terminus of ZZ, and the other linker was fused to the N-terminus of AP, Luc, or GOD, respectively. The resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged ZZ and AP, Luc, or GOD were site-specifically conjugated by SrtA through the extra peptidyl linkers in vitro. The SrtA reaction had little influence on either the antibody-binding activity of the ZZ moiety or the enzymatic activity of AP, Luc, or GOD moieties of the conjugates. Additionally, antibody-ZZ-proteins were yielded easily by mixing antibody with ZZ-AP, ZZ-Luc, or ZZ-GOD, allowing their use in an enzyme-linked immunosorbent assay. These results suggest that the enzymatic approach with SrtA facilitates the construction of ZZ-proteins. Furthermore, mixing antibody and ZZ-proteins produces a wide variety of antibody-ZZ-proteins.

INTRODUCTION Site-specific protein modification is a powerful strategy in biological research, because it enables protein manipulation without significant loss of function. Chemical modification is widely employed, although this strategy is not always “sitespecific”, but rather “residue-specific”. If the target protein has a number of reactive residues, chemically modified products are often heterogeneous due to random modifications. Since cysteine (Cys) residues do not occur frequently in native proteins and a genetically introduced Cys may represent the only site to be modified, Cys-based protein modification with thiol-selective reagents has often been employed (1-4). Native chemical ligation, such as N-terminally introduced Cys-specific modification, is a more sophisticated strategy due to its being N-terminal-specific (5, 6). One obstacle is the difficulty associated with producing Cys-introduced proteins due to the formation of inclusion bodies or incorrect disulfide bridges. Enzymatic approaches to site-specific protein modification have attracted much attention because the substrate specificity of an enzyme enables “site-specific” protein modification (7, 8). Several kinds of transglutaminases have been used for protein modification with primary amine-containing molecules such as fluorescence probes or PEG (9-11). Briefly, a short substrate sequence for transglutaminase is genetically introduced at the N- or C-terminus of the target protein. The expressed protein is modified with a small primary amine-containing molecule. Sortase has also been used for protein modification (12-16). The most studied one is Sortase A (SrtA), a transpeptidase from Staphylococcus aureus. Wild-type SrtA consists of 206 amino acids (23.5 kDa). N-Terminal 59-amino-acid truncated SrtA (17 kDa and pI ) 6.8) has been used in many studies (13-16). SrtA recognizes the LPXTG sequence, cleaves between the Thr * Corresponding author. Tsutomu Tanaka (T. Tanaka), E-mail: [email protected]. Tel/Fax: +81-78-803-6202. † Department of Chemical Science and Engineering, Graduate School of Engineering. ‡ Organization of Advanced Science and Technology.

and Gly residues, and subsequently links the carboxyl group of Thr to an amino group of N-terminal glycine oligomers by a native peptide bond (17, 18). Recombinant soluble SrtA has been used for peptide-protein or protein-protein ligation in vitro because of its greater substrate specificity (19-21). The advantages of this enzymatic protein modification strategy are that it is highly selective and mild compared to conventional methods. Antibody-protein conjugates have been widely used in various areas because of their remarkable molecular recognition properties. Generally, a Cys-based modification strategy is employed; however, site-specific modification of antibodies is not always achieved and the yield of modified antibody is not yet high enough. Although short peptide tag sequences should be introduced into antibodies in order to apply the enzymatic modification strategy, the establishment of a recombinant antibody production system is too difficult and is less versatile (22-24). Here, we demonstrate site-specific antibody-protein conjugation using SrtA. We focused on an antibody-binding domain called the ZZ domain. The ZZ domain has high affinity to the Fc region of various kinds of antibodies, and its genetic manipulation and mass production is very easy (25, 26). The strategy of this study is shown in Figure 1A. ZZ domains were site-specifically conjugated with other proteins using sortase, and then the desired antibody-ZZ-protein conjugates were created by simply mixing the antibodies and ZZ-protein conjugates. We genetically introduced the LPETG sequence to the C-terminus of a ZZ domain (ZZ-LPETG). As model of partner proteins, alkaline phosphatase (27), luciferase (28, 29), and glucose oxidase (30) were used (Figure 1B). Pentaglycine appended alkaline phosphatase (Gly5-AP), luciferase (Gly5Luc), and triglycine appended glucose oxidase (Gly3-GOD) were prepared (Figure 1B). ZZ-LPETG and Gly5-AP were sitespecific conjugated by a SrtA-mediated reaction. In addition, Gly5-Luc and Gly3-GOD were also conjugated with ZZ-

10.1021/bc100206z  2010 American Chemical Society Published on Web 11/11/2010

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Figure 1. (A) Schematic illustration of antibody-ZZ-protein conjugation using Sortase A. ZZ-LPETG and (Gly)n-protein are site-specifically conjugated by Sortase A. The resultant ZZ-protein is simply mixed with whole antibody, and antibody-ZZ-protein is obtained due to the affinity between Fc region of ZZ and Fc region of antibody. (B) N-Terminal glycine appended proteins used in this study. Alkaline phosphatase and glucose oxidase were expressed with pelB signal sequence and Taka amylase (TAA) signal sequence, respectively. The moiety of signal sequence was cleaved by signal peptidase in each host cells; the N-terminal glycine exposed proteins were obtained. In the case of luciferase, Factor Xa cleavage between thioredoxin and luciferase could expose the N-terminal glycine of luciferase.

LPETG. Finally, we successfully demonstrated target molecule detection using antibody-ZZ-protein conjugates.

EXPERIMENTAL PROCEDURES Plasmid Construction. The plasmid for pentaglycine appended alkaline phosphatase expression was constructed as follows. The gene encoding alkaline phosphatase (AP) from E. coli was amplified by PCR using E. coli genomic DNA as a template with the following primers: 5′-C CGT CAT ATG AAA TAC CTG CTG CCG ACC GCT GCT GCT GGT CTG CTG CTC CTC GCT GCC CAG CCG GCG ATG GCC GGC GGT GGA GGT GGA TCC ACA CCA GAA ATG CCT GTT CTG GAA AAC C-3′ and 5′-TTA ACT CGA GTG CGG CCG CTT TCA GCC CCA GAG CGG CTT TCA TGG-3′. (pelB leader sequence is underlined.) The amplified fragment was subcloned into the NdeI/XhoI sites of pET22b (Novagen). The resultant plasmid was named pET22b-Gly5-AP. The pelB leader sequences were cleaved during expression (31), and N-terminal glycine was exposed (Figure 1B). The plasmid for LPETG sequence appended ZZ domain expression was constructed as follows. The gene encoding ZZ was amplified by PCR using pGLDLd33-ZZ (32) as a template using the following primers: 5′-GC GGT ACC GGA TCC GGT ATT GAG GGT CGC GGC GGT GGA GGT GGT GCG CAA CAC GAT GAA GCC GTA GAC-3′ and 5′-GC GAG CTC CTA GAA TTC ACC GCC AGT CTC AGG CAG GCC GCC GCC GGA GGA CTC TTT CGG CGC CTG AGC ATC ATT TAG C-3′. The amplified fragment was ligated into the KpnI/ SacI sites of pBAD-Gly5-EGFP (15). The resultant plasmid was named pBAD-ZZ-LPETG. The plasmid for pentaglycine appended luciferase (Luc) expression was constructed as follows. The gene encoding Luc was amplified by PCR using pCBG68Basic Vector (Promega) as a template using the following primers: 5′-GCG GTA CCG GAT CCG GTA TTGAGG GTC GCG GCG GTG GAG GTG GTG ACT ACA AGG ATG ACG ACG ACA AGA TGG TGA AAC GCG AAA AGA ACG TGA TCT ACG G-3′ and 5′-GCG AGC TCC TAG CCG CCA GCT TTT TCG AGG AGT TGC TTC AGC-3′. The amplified fragment was ligated into the KpnI/SacI sites of pBAD-Gly5EGFP (15). The resultant plasmid was named pBAD-Gly5-Luc. The plasmid for triglycine appended glucose oxidase (GOD) expression was constructed as follows. The gene encoding GOD was amplified by PCR using Aspergillus niger genomic DNA as a template using the following primers: 5′-GC CTC GAG

AAA AGA GGC GGT GGA CAC CAC CAC CAC CAC CAC AGC AAT GGC ATT GAA GCC AGC CTG GTG ACT GAT CCC-3′ and 5′-G CAT AGC GGC CGC TCA CTG CAT GGA AGC ATA ATC TTC CAA-3′. The second PCR was carried out using the amplified fragment as a template using the following primers: 5′-G CAT ACT AGT AAA AGA GGC GGT GGA CAC CAC CAC CAC CAC CAC-3′ and 5′-G CAT AGC GGC CGC TCA CTG CAT GGA AGC ATA ATC TTC CAA3′. The amplified fragment was ligated into the SpeI/NotI sites of pISI-EGFP (33). The resultant plasmid was named pISI-Gly3GOD. In the case of Gly3-GOD, secretion signal sequence derived from taka amylase (33) were cleaved during secretion, and N-terminal glycine was exposed (Figure 1B). The plasmid for myc sequence appended luciferase (Luc) expression was constructed as follows. The gene encoding Luc was amplified by quick change using the pBAD-Gly5-Luc Vector as a template using following primers: 5′-GAC TAC AAG GAT GAC GAC GAC AAG GAA CAA AAG TTG ATC AGC GAA GAG GAC CTC ATG GTG AAA CGC GAA AAG AAC GTG-3′ and 5′-CAC GTT CTT TTC GCG TTT CAC CAT GAG GTC CTC TTC GCT GAT CAA CTT TTG TTC CTT GTC GTC GTC ATC CTT GTA GTC-3′. The resultant plasmid was named pBAD-Gly5-myc-Luc. The plasmid for pentaglycine and FLAG sequence appended red fluorescence protein (RFP) expression was constructed as follows. The gene encoding RFP was amplified by PCR using pmRFP-N1 (Clontech) as a template using the following primers: 5′-CGG GGT ACC ATT GAG GGT CGC GGC GGT GGA GGT GGT AGC GAT TAC AAG GAT GAC GAC GAT AAG AGC GAC AAC ACC GAG GAC GTC ATC AAG GAG TTC-3′ and 5′-GAG CTC CTA GCT AGC ATA ATC TGG AAC ATC ATA TGG ATA GCT GGA GCC GGA GTG GCG GGC CTC GGC GTG CTC-3′. The amplified fragment was ligated into the KpnI/SacI sites of pBAD-Gly5-EGFP (15). The resultant plasmid was named pBAD-Gly5-FLAG-RFP. Protein Expression and Purification. SrtA and green fluorescence protein (GFP) were expressed and purified according to a previous report (15). The expression and purification of Gly5-AP was conducted as follows. Plasmid pET22b-Gly5AP was transformed into E. coli BL21 (DE3) (Novagen). The cells were grown in LB medium to an OD (600 nm) value of 0.8, at which time expression of protein was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. After growth for an additional 16 h

Enzyme-Mediated Antibody-Protein Modification

at 25 °C, the cells were harvested by centrifugation. The cell pellets were resuspended in 50 mM Tris-HCl, 300 mM NaCl, pH 7.5, and lysed by sonication. Gly5-AP was purified from the soluble fraction of the lysate by TALON metal affinity resins (Clontech) according to the manufacturer’s protocol and dialyzed against 50 mM Tris-HCl, 300 mM NaCl, pH 7.5. Protein concentration was determined using a BCA protein assay kit (Pierce). Plasmids pBAD-ZZ-LPETG, pBAD-Gly5-Luc, pBAD-Gly5myc-Luc, and pBAD-Gly5-FLAG-RFP were induced in E. coli Top10 (Invitrogen) by the addition of 0.1% L-arabinose and subsequent incubation for 16 h at 25 °C. ZZ-LPETG, Gly5Luc, Gly5-myc-Luc, and Gly5-FLAG-RFP were purified using TALON metal affinity resin as described above. In the case of Gly5-Luc, Factor Xa (New England Biolabs) cleavage was carried out according to manufacturer’s procedure to expose the N-terminal glycine (Figure 1B). After cleavage, Factor Xa was removed by Benzaimidine Sepharose 4 Fast Flow (GE Healthcare), and the cleaved thiredoxin moiety was also removed by TALON metal affinity resin. The Aspergillus oryzae niaD mutant (strain IF4), derived from wild-type A. oryzae, OSI1031, was used as the GOD expression host. Czapek-Dox (CD) medium plates (2% glucose, 0.3% NaNO3 (CD-NO3), 0.2% KCl, 0.1% KH2PO4, 0.05% MgSO4 · 7H2O, and 0.8 M NaCl, pH 6.0) containing 1.5% agar were used as the minimal medium to select the fungal transformants. GPY medium (3% glucose, 0.2% KCl, 0.1% KH2PO4, 0.05% MgSO4 · 7H2O, 1% peptone, and 0.5% yeast extract, pH 6.0) was used for growing the transformants and for GOD expression. The transformation of A. oryzae was carried out according to the method described by Gomi et al. (1987). The resultant transformants were subcultured on a CDNO3 medium plate three times for obtaining the stable expression transformants. Then, the transformants were seeded on 250 mL of GPY medium and cultivated for 6 days at 30 °C. The culture supernatants were separated using Mira cloth (Millipore), and the secreted GOD was purified using TALON metal affinity resin according to the manufacturer’s procedure. SrtA Reaction between ZZ-LPETG and Pentaglycine Appended Proteins. The reaction between ZZ-LPETG (10 µM) and Gly5-AP (25 µM), Gly5-Luc (10 µM), and Gly3-GOD (10 µM) was initiated by the addition SrtA (35 µM). The reaction was carried out in 20 mM Tris-HCl, 150 mM NaCl, 0.5 mM CaCl2, pH 7.5 at 24 °C. After incubation for 15 h, the reaction was terminated by mixing with SDS-PAGE sample buffer (50 mM Tris-Cl, 2% SDS, 6% 2-mercaptoethanol) and boiling. The samples were then subjected to SDS-PAGE, and the gels were stained with Coomassie brilliant blue R-250 (Nacalai Tesque). AP Activity Analysis. In order to evaluate AP activity, luminescence was determined using CDP-Star (Applied Biosystems) after the SrtA reaction. Gly5-AP (25 µM), ZZ-LPETG (10 µM), and SrtA (35 µM), creating a ZZ-AP conjugate, were placed in each well of a 96-well microplate. After the addition of CDP-Star, the luminescence intensity in each well was measured at room temperature with a Wallac 1420 ARVOsx Multi Label Counter (Perkin-Elmer). Luciferase Activity Analysis. In order to evaluate Luc activity, the luminescence was determined using Bright-Glo (Promega) after the SrtA reaction. Gly5-Luc (10 µM), ZZLPETG (10 µM), and SrtA (35 µM), creating a ZZ-Luc conjugate, were placed in each well of a 96-well microplate. After the addition of Bright-Glo, the luminescence intensity in each well was measured at room temperature with a Wallac 1420 ARVOsx Multi Label Counter. GOD Activity Analysis. In order to evaluate GOD activity, the absorbance was determined using glucose and peroxidase and a coloring reagent (Nacalai Tesque) after the SrtA reaction.

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Gly3-GOD (10 µM), ZZ-LPETG (10 µM), and SrtA (35 µM), creating a ZZ-GOD conjugate, were placed in each well of a 96-well microplate. After the addition of glucose (0.5 M), peroxidase (1 mg/mL), and coloring reagent (100 µL), the absorbance at 450 nm in each well was measured at room temperature with a Wallac 1420 ARVOsx Multi Label Counter. ELISA Using Antibody-ZZ-Protein Conjugates. BSA was Gly5-FLAG-RFP (10 µg/mL in PBS) were placed in each well of a 96-well microplate and incubated for 15 h at 4 °C to allow adsorption to the surface of the wells. At the same time, 15 µM ZZ-AP conjugate and 6 µM ZZ-Luc conjugate were added with anti-FLAG antibody 20 µg/mL (Sigma) and incubated for 1 h at 4 °C. Each well was washed three times with PBS. Then, anti-FLAG antibody-ZZ-AP conjugate and anti-FLAG antibodyZZ-Luc conjugate were added to each well and incubated for 1 h at 4 °C, followed by washing three times with PBS. After the addition of CDP-Star (Applied Biosystems) and Bright-Glo (Promega), the luminescence intensity in each well was measured using a Wallac 1420 ARVOsx Multi Label Counter. BSA or Gly5-myc-Luc (10 µg/mL in PBS) was placed in each well of a 96-well microplate and incubated for 15 h at 4 °C to allow adsorption to the surface of the wells. At the same time, 6 µM ZZ-GOD conjugate was added with antimyc antibody 20 µg/mL (Bethyl Laboratories) and incubated for 1 h at 4 °C. Each well was washed three times with PBS. Then, antimyc antibody-ZZ-GOD was added to each well and incubated for 1 h at 4 °C, followed by washing three times with PBS. After the addition of glucose, peroxidase, and coloring reagent (Nacalai Tesque), the absorbance at 450 nm in each well was measured using a Wallac 1420 ARVOsx Multi Label Counter. In order to evaluate the applicability of the ZZ-Luc conjugate, ELISA for GFP was carried out. Different amounts of GFP (0, 0.25, 0.5, 2.5, 5, 6.25, 12.5, 25 nM in PBS) were placed in each well of a 96-well microplate and incubated for 15 h at 4 °C to allow adsorption to the surface of the wells. At the same time, 6 µM ZZ-Luc conjugate was added with anti-GFP antibody 20 µg/mL (Medical & Biological Laboratories) and incubated for 1 h at 4 °C. Each well was washed three times with PBS. Then, anti-GFP antibody-ZZ-Luc conjugate was added to each well and incubated for 1 h at 4 °C, followed by washing three times with PBS. After the addition of Bright-Glo, the luminescence intensity in each well was measured using a Wallac 1420 ARVOsx Multi Label Counter.

RESULTS AND DISCUSSION Site-Specific Conjugation between ZZ-LPETG and Pentaglycine Appended Proteins Using SrtA. Figure 1A shows the effect of SrtA treatment on ZZ-LPETG and Gly5AP. When Gly5-AP, ZZ-LPETG, and SrtA were mixed, a ZZAP conjugate (ca. 75 kDa) was clearly observed (Figure 2A, lane 7). ZZ-LPETG and Gly5-AP themselves were not susceptible to SrtA treatment (Figure 2A, lanes 5, 6), In addition, when ZZ-LPETG and AP without the pentaglycine tag at its Nterminus were mixed, the ZZ-AP conjugate was not observed, in spite of SrtA treatment (Figure 2B). These results suggest that site-specific ZZ-LPETG and Gly5-AP conjugation were carried out through the tag sequences. The yield of ZZ-AP was about 65% of the Gly5-AP, as evaluated by the ratio of the Gly5-AP band intensity before conjugation (Figure 2A, lane5) to that after conjugation (Figure 2A, lane 7). The ZZ-AP conjugates were also stable for more than one week even in the presence of the Sortase A, which is confirmed by SDSPAGE (data not shown). To expand the versatility of this strategy, we carried out sitespecific protein conjugation between ZZ-LPETG and other proteins. Firefly luciferase and glucose oxidase were employed

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Figure 2. SDS-PAGE analysis of the site-specific reaction products after Sortase A treatment: (A) lane 1, Gly5-AP; lane 2, ZZ-LPETG; lane 3, Sortase A; lane 4, Gly5-AP; and ZZ-LPETG; lane 5, Gly5-AP and Sortase A; lane 6, ZZ-LPETG and Sortase A; lane 7, Gly5-AP and ZZ-LPETG and Sortase A. (B) lane 1, AP; lane 2, ZZ-LPETG; lane 3, Sortase A; lane 4, AP and ZZ-LPETG; lane 5, AP and Sortase A; lane 6, ZZ-LPETG and Sortase A; lane 7, AP and ZZ-LPETG and Sortase A.

Figure 3. SDS-PAGE analysis of the reaction products after Sortase A treatment. (A) lane 1, Gly5-Luc; lane 2, ZZ-LPETG; lane 3, Sortase A; lane 4, Gly5-Luc and ZZ-LPETG; lane 5, Gly5-Luc and Sortase A; lane 6, ZZ-LPETG and Sortase A; lane 7, Gly5-Luc and ZZ-LPETG and Sortase A. (B) lane 1, Gly3-GOD; lane 2, ZZ-LPETG; lane 3, Sortase A; lane 4, Gly3-GOD and ZZ-LPETG; lane 5, Gly3-GOD and Sortase A; lane 6, ZZ-LPETG and Sortase A; lane 7, Gly3-GOD and ZZ-LPETG and Sortase A.

as reporter proteins. Firefly luciferase is widely used as a detection enzyme in ELISA, as well as in reporter gene assays. Luciferase was expressed as a thioredoxin-tag fusion protein using E. coli as a host. Then, N-terminal pentaglycine was exposed by protease (Factor Xa) digestion, and the thioredoxin tag was removed. Glucose oxidase is also widely used as a detection enzyme due to its high sensitivity against glucose. However, glycosylation is required for GOD activity, and hence, GOD cannot be expressed using E. coli. Other hosts such as Aspergillus oryzae or yeast are required. Figure 3A shows the effect of SrtA treatment of ZZ-LPETG and Gly5-Luc. Gly5-Luc and Gly3-GOD were successfully expressed and purified using appropriate hosts. The smeared bands of GOD were caused by glycosylation. As expected, the ZZ-Luc conjugate (ca. 100 kDa) was successfully obtained after SrtA treatment (Figure 3A, lane 7). Figure 3B shows the effect of SrtA treatment of ZZ-LPETG and Gly3-GOD. The ZZ-GOD conjugate (ca. 110 kDa) was also successfully obtained after SrtA treatment (Figure 3B, lane 7). Neither Luc nor GOD was susceptible to SrtA because of its high substrate specificity. The yield of ZZ-GOD conjugates estimated with Gly3-GOD band intensity was about 65%, suggesting that the reactivity of SrtA

was not affected by the target protein and/or protein modification such as glycosylation. Evaluation of Enzymatic Activity after the Sortase Reaction. Figure 4 shows the enzymatic activity after SrtA treatment. The enzymatic activity of AP was almost the same when Gly5-AP was mixed with ZZ-LPETG or SrtA (Figure 4A, columns 1, 4, 5). This clearly shows that AP activity was not affected by other proteins. After SrtA treatment, the produced ZZ-AP conjugate retained its activity at almost the same level as Gly5-AP (Figure 4A, column 7). The other conjugates, ZZ-Luc and ZZ-GOD, both had enzymatic activity after SrtA treatment (Figure 4B,C, columns 1, 7). These data clearly show that SrtA enables conjugation of proteins without significant loss of their functions. Several previous reports have shown that protein activity after enzymatic conjugation is retained (10-16). Our results are consistent with these reports and demonstrate the superiority of the enzymatic protein conjugation method. Detection of Target Molecules Using AntibodyZZ-Protein Conjugates. Encouraged by these findings, we carried out the detection of target molecules using antibodyZZ-protein conjugates. A moiety of the FLAG tag sequence in

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Figure 4. Evaluation of the enzymatic activity after Sortase treatment. (A) Alkaline phosphatase activity: column 1, Gly5-AP; column 2, ZZLPETG; column 3, Sortase A; column 4, Gly5-AP and ZZ-LPETG; column 5, Gly5-AP and Sortase A; column 6, ZZ-LPETG and Sortase A; column 7, Gly5-AP and ZZ-LPETG and Sortase A. (B) Luciferase activity: column 1, Gly5-Luc; column 2, ZZ-LPETG; column 3, Sortase A; column 4, Gly5-Luc and ZZ-LPETG; column 5, Gly5-Luc and Sortase A; column 6, ZZ-LPETG and Sortase A; column 7, Gly5-Luc and ZZLPETG and Sortase A.

Gly5-FLAG-RFP was employed as a model target molecule. ZZ-AP and anti-FLAG antibody (whole IgG) were simply

mixed, and an anti-FLAG antibody-ZZ-AP conjugate was produced. As a negative control, BSA was employed. As shown

Figure 5. FLAG-tag detection using ZZ-enzyme conjugate. (A) ZZ-AP: column 1, Gly5-AP; column 2, ZZ-LPETG; column 3, Sortase A; column 4, Gly5-AP and ZZ-LPETG; column 5, Gly5-AP and Sortase A; column 6, ZZ-LPETG and Sortase A; column 7, Gly5-AP and ZZ-LPETG and Sortase A. (B) ZZ-Luc: column 1, Gly5-Luc; column 2, ZZ-LPETG; column 3, Sortase A; column 4, Gly5-Luc and ZZ-LPETG; column 5, Gly5Luc and Sortase A; column 6, ZZ-LPETG and Sortase A; column 7, Gly5-Luc and ZZ-LPETG and Sortase A. myc-tag detection using ZZ-enzyme conjugate. (C) ZZ-GOD: column 1, Gly3-GOD; column 2, ZZ-LPETG; column 3, Sortase A; column 4, Gly3-GOD and ZZ-LPETG; column 5, Gly3-GOD and Sortase A; column 6, ZZ-LPETG and Sortase A; column 7, Gly3-GOD and ZZ-LPETG and Sortase A.

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quantum dot (14-16). Also notable is that the additional short sequences (i.e., LPETG or GGGGG) may have little effect on the protein expression and refolding, which is possible to allow the ligation of ZZ-LPETG to tag-introduced proteins after refolding. Our strategy is highly useful and versatile for producing antibody-protein conjugates.

ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Young Scientist B (21760638) of Japan Society for the Promotion of Science (JSPS) and Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe), MEXT, Japan.

LITERATURE CITED Figure 6. ELISA with anti-GFP antibody-ZZ-Luc conjugate. The signal intensities of wells containing varying concentrations of the antigen, GFP, were measured. Data are averages from three independent experiments and SD are indicated.

in Figure 5A, Gly5-FLAG-RFP was successfully detected using the anti-FLAG antibody-ZZ-AP conjugate (Figure 5A, column 7). No signal was observed against BSA, and no signal was observed without the ZZ-AP conjugate or by using a mixture of ZZ and AP (Figure 5A, columns 1-6). We also utilized the anti-FLAG antibody to ZZ-Luc conjugate. As shown in Figure 5B, Gly5-FLAG-RFP was successfully detected using the antiFLAG antibody-ZZ-Luc conjugate (Figure 5B, column 7). No signal was observed against BSA, and no signal was observed without the ZZ-Luc conjugate (Figure 5B, columns 1-6). The S/N (signal-to-noise) ratio was 40 in the presence or absence of Gly5-FLAG-RFP, which is 7-fold higher than that of ZZ-AP. This is due to the higher activity of luciferase compared to AP. Additionally, Gly5-myc-Luc was employed as a model target molecule. ZZ-GOD and antimyc antibody (whole IgG) were simply mixed and antimyc antibody-ZZ-GOD conjugates were produced. As a negative control, BSA was employed. As shown in Figure 5C, Gly5-myc-Luc was successfully detected using the antimyc antibody-ZZ-GOD conjugate (Figure 5C, column 7). No signal was observed against BSA, and no signal was observed without the ZZ-GOD conjugate (Figure 5C, columns 1-6). ELISA was also carried out to test the applicability of antibody-ZZ-Luc conjugate as immunoassay reagent. As shown in Figure 6, signal intensity was increased in response to the concentration of GFP administrated, and the detection limit of GFP with anti-GFP antibody-ZZ-Luc conjugates was found to be around 2.5-5 nM. In the case of using anti-HA antibodyZZ-Luc conjugate, no signal observed against GFP and no signal was increased in response to the GFP concentration, as expected (data not shown). These results showed that antibody-ZZ-protein conjugate is able to be utilized for immunoassay because of its bifunctionality. In conclusion, ZZ-LPETG and some enzymes were sitespecifically conjugated by a SrtA-mediated reaction. These conjugates retained both the enzymatic activity of AP, Luc, or GOD and the IgG binding activity of ZZ after SrtA treatment. Because genetic engineering of the whole antibody is very difficult, ZZ-protein conjugate can be one of the ways to produce a bifunctional antibody by only mixing with whole antibody. Sortase A also can ligate between proteins and functional small molecules site-specifically (13-16), suggesting the extension of this ZZ-protein conjugate to ZZ-functional molecule conjugate, for example, fluorescent dyes and/or

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