Creation of a Ligand-Dependent Enzyme by ... - ACS Publications

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Creation of a Ligand-Dependent Enzyme by Fusing Circularly Permuted Antibody Variable Region Domains Hiroto Iwai,† Miki Kojima-Misaizu,† Jinhua Dong,‡ and Hiroshi Ueda*,‡ †

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, Kanagawa 226-8503, Japan

‡

S Supporting Information *

ABSTRACT: Allosteric control of enzyme activity with exogenous substances has been hard to achieve, especially using antibody domains that potentially allow control by any antigens of choice. Here, in order to attain this goal, we developed a novel antibody variable region format introduced with circular permutations, called Clampbody. The two variable-region domains of the antibone Gla protein (BGP) antibody were each circularly permutated to have novel termini at the loops near their domain interface. Through their attachment to the N- and C-termini of a circularly permutated TEM-1 β-lactamase (cpBLA), we created a molecular switch that responds to the antigen peptide. The fusion protein specifically recognized the antigen, and in the presence of some detergent or denaturant, its catalytic activity was enhanced up to 4.7-fold in an antigen-dependent manner, due to increased resistance to these reagents. Hence, Clampbody will be a powerful tool for the allosteric regulation of enzyme and other protein activities and especially useful to design robust biosensors.

cpBLA), respectively. However, although the results proved the principle of antigen-dependent enzyme regulation and the antigen-dependent growth of E. coli in the presence of ampicillin,16 the specific activity only increased up to 25%, which was not sufficient to justify its further application as a biosensor. In order to effectively control the catalytic activity of such artificial allosteric enzymes, it is crucial to structurally optimize each domain, including the linker connecting the two evolutionally distinct proteins. In the case of protein-fragment complementation assay (PCA), the protein-fragment complementarity is effectively induced when the protein fragments come close together in the correct orientation. In the case of circular permutation, proteins also seem to require precise control of the distance and orientation of their termini for catalytic regulation. For example, Wright, Ke, and colleagues recently reported the NMR and crystal structures of a fusion protein comprising cpMBP and cpBLA.17,18 In these reports, the structure of the connection sites between cpMBP and cpBLA was locally disordered but less perturbed after the binding of maltose to the cpMBP domain, indicating their pivotal role on the catalytic activities of the enzymes. Until now, polypeptide linkers have been widely used to connect antibodies to other proteins. However, the naive insertion of a flexible linker often leads to insufficient signal transduction from the binding domain to the catalytic domain.

Antibodies recognize a wide variety of molecules with high affinity and specificity, and they have been successfully applied to a range of fields such as immunoassays and therapeutics. As the recognition module of an antibody, the variable region (Fv) presents a stable scaffold composed of structurally conserved framework regions and highly divergent complementaritydetermining regions. To date, many Fvs have been the target of protein engineering and are fused with other molecules or proteins to create novel molecular probes,1−5 sensor proteins,6−8 and fusion enzymes for prodrug therapy.9−11 Among the range of applications, the molecular switch comprising an enzyme and antibody domains is considered highly useful, owing to the recognition specificity derived from the antibody and the efficient catalytic activity from the enzyme. To date, there have been a number of reports on such fusion proteins, whose binding signal is transduced and converted to a change in catalytic activity. For example, the catalytic activity of two fusion proteins, each consisting of an antibody variable region and a truncated β-galactosidase fragment, was regulated through the reconstitution of the enzyme fragments induced by the antigen-Fv complex formation and applied to the sensitive homogeneous immunoassay of small haptens.12 Another example is a fusion protein consisting of a circularly permutated TEM-1 β-lactamase (cpBLA) and Fv.13 By use of cpBLA, which was originally reported as a part of single-molecule maltose-sensing switch that was fused with a circularly permutated maltose binding protein (cpMBP),14,15 a fusion protein that could detect antigens was created by linking the native variable domains VH and VL to the N terminus and C terminus of cpBLA (Fv© XXXX American Chemical Society

Received: January 22, 2016 Revised: February 23, 2016

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DOI: 10.1021/acs.bioconjchem.6b00040 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. Creation of Clampbody and its fusion to cpBLA (a) Schematic structure of single chain Clampbody (sc-Cbody). cpVH and cpVL chains are shown in magenta and cyan, respectively. The (G4S)3 linkers connecting their native N- and C-termini are drawn as dashed lines. Two cysteine residues were inserted at the N- and C-termini of cpVH to promote correct folding via disulfide linkage. The SG4 linker connecting cpVH and cpVL is drawn as dotted line. (b) Schematic structure of Cbody-cpBLA, in which the structure of cpBLA (PDB code 4DXB) is shown in green. (c, d) Expression and purification of Cbody-cpBLA. (c) CBB-stained SDS−PAGE. (1) MW marker, (2) concentrated culture supernatant, (3) soluble fraction from the cell lysate, (4) insoluble fraction from the same, (5) solubilized insoluble protein, (6) purified and refolded protein. (d) Western blot to detect the His tag. The lanes are the same as in (c). The bands for Cbody-cpBLA are indicated by an arrow.

The distance between the 3 and 3b loops of VH and VL of KTM219 was estimated to be approximately 12.3 Å by homology modeling. Therefore, to design novel antibody domains that have their N- and C-termini in these loops, we performed following circular permutations; for circularly permutated VH (cpVH), the novel termini were generated between Pro41H and Gly42H (according to Kabat numbering scheme), and the native N- and C-termini were connected via a flexible linker (G4S)3. Similarly, the circularly permutated VL (cpVL) was designed by generating termini between Pro40L and Gly41L. Moreover, as the VH fragment is often less stable than the paired VL fragment in many antibodies, two cysteine residues were inserted at the N- and C-termini of cpVH to promote the correct folding and increased stability through disulfide linkage (Figure 1a). First, we investigated whether this novel Fv format, termed Clampbody, can recognize its antigen peptide by creating a single chain protein where cpVH and cpVL are connected through the short linker (SG4) (single chain Clampbody; sc-Cbody). To this end, E. coli SHuffle T7 Express lysY cells were transformed with the expression vector harboring the gene for sc-Cbody, and the expression of this fusion protein was investigated for in the culture supernatant, the intracellular soluble fraction, and the insoluble fraction. According to the SDS−PAGE and Western blot using the HRP-anti-His-tag antibody, the sc-Cbody was detected mostly in the insoluble fraction. Therefore, we purified and refolded scCbody from the solubilized insoluble protein using immobilized metal affinity chromatography and stepwise dialysis to introduce correct disulfide linkages (Figure S1 in Supporting Information) as described earlier.21 The antigen-binding

Here, we searched for a structurally tighter connection between the antibody and cpBLA. The distance between the N- and Ctermini of ordinary VH and VL is between 30 and 40 Å, and this distance is considered too long for controlling the orientation of protein fragments or circularly permutated enzymes. Hence, we focused on the 3 and 3b loops located near the domain interface between VH and VL (Figure 1a). These loops have been used as a connection site to create a unique single-chain antibody format (permutated Fv; pFv), indicating its high structural tolerance.19 By extending this idea, we designed doubly circularly permutated antibody variable domains (Clampbody; Cbody) that have novel termini at these positions. After this, we connected the Clampbody to the cpBLA and investigated the specific control of enzyme activity by the antigen.

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RESULTS AND DISCUSSION

We applied the antibody KTM219, which recognizes the Cterminal region of the human bone Gla protein (BGP),20 to circular permutations. BGP is also known as osteocalcin and is one of the major bone-derived serum proteins secreted by osteoblasts. The concentration of BGP in healthy human serum is between 1 and 2 nM. However, the level increases in some patients with endocrine or bone disorders such as hyperparathyroidism, hyperthyroidism, Paget’s disease, and osteoporosis. Therefore, the serum level of BGP can be used as a biomarker for these diseases, and the development of a handy assay for detecting this protein should be useful in clinical application. B


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expressed as inclusion bodies using SHuffle T7 Express lysY cells. On the basis of the purification methods of sc-Cbody, we successfully purified and refolded Cbody-cpBLA (Figure 1c,d). The antigen binding activity of Cbody-cpBLA was confirmed by ELISA using immobilized biotinylated BGP-C11 peptide. The fusion protein clearly showed a specific binding to biotinylated antigen while showing its negligible binding to streptavidin (Figure 2b). This result indicates that the new recognition unit Cbody was successfully used to make an antibody−enzyme fusion protein. We then investigated the catalytic activity of Cbody-cpBLA. In phosphate buffered saline (PBS), Cbody-cpBLA hydrolyzed the fluorogenic substrate fluorocillin, and the catalytic activity showed modest increase in the presence of 1 μg/mL BGP-C10 peptide (p ≈ 0.02) (Figure 3a). From this result, we reasoned that Cbody-cpBLA was somewhat stabilized by the binding of the antigen peptide and showed a property as an allosteric enzyme. Since Cbody-cpBLA also showed a relatively high catalytic activity in the absence of the antigen, next we tried to reduce this background catalytic activity in order to increase its antigen dependency. To this end, we tested adding several denaturants and detergents, namely, urea, Triton X-100, and Tween-20, to the reaction buffer. In these conditions, the catalytic activity of Cbody-cpBLA in the absence of the antigen was considerably lowered. However, in the presence of the antigen, the activity was relatively maintained, which resulted in the significantly increased antigen-dependency in catalytic activity (p < 0.01) (Figure 3b,c and SI Figure S2). Classically, the allosteric regulation of enzymes has been considered as a structural or conformational change of catalytic domain or subunits. More recently, however, thermodynamic properties or protein stability are also considered to drive allosteric regulation.23 Choi and colleagues also reported that the stability and catalytic activity of cpBLA fused with MBP was regulated by some denaturant, reducing agent, and pH in a ligand-dependent manner.24−26 We think that our study also demonstrates this novel type of allosteric regulation. Then we investigated the dose response and recognition specificity of Cbody-cpBLA at the optimized reaction condition. In PBS containing 0.1% Triton X-100 and 250 μg/mL bovine serum albumin (BSA), the catalytic activity of Cbody-cpBLA showed a clear dose dependency to BGP-C10. On the other hand, it did not show any response to BGP-C10dV, a C-terminally

activity of sc-Cbody was confirmed using ELISA, which showed sc-Cbody specifically binding to the immobilized BGP-C11 antigen peptide (Figure 2a). The EC50 was 23.0 ± 1.2 nM and

Figure 2. Antigen binding activity of Fv, sc-Cbody, and Cbody-cpBLA. (a) Binding of parental Fv (red triangle) and sc-Cbody (blue circle) to the biotinylated BGP-C11 antigen immobilized at the indicated concentrations. (b) Binding of Cbody-cpBLA to the biotinylated BGPC11 immobilized at 0.9 μM. Blue and red bars indicate the binding signals to antigen-positive and negative wells, respectively. Averages of three samples with an error bar of 1 SD are shown.

was approaching that of the mixture of parental VH and VL (Fv, EC50 = 3.6 ± 0.3 nM).22 However, the signal intensity of scCbody at high antigen concentration was smaller than that of Fv, indicating that a fraction of Cbody was not completely refolded and the binding activity of sc-Cbody might be still underestimated. Considering these observations, these data indicate that sc-Cbody retains the antigen recognition activity of the parental Fv, although its antigen binding activity is slightly reduced. In order to attain the regulation of enzyme activity by Clampbody, we designed a fusion protein comprising Clampbody and cpBLA. The schematic structure of this fusion protein, Cbody-cpBLA, is shown in Figure 1b. We used the cpBLA reported by Guntas et al.14 According to the crystal structure of this MBP-cpBLA (PDBcode 4DXB),18 we constructed a model of Cbody-cpBLA by connecting Clampbody and cpBLA in a similar manner to MBP-cpBLA. On the basis of this structure, cpVH and cpVL were connected to the Nand C-termini of cpBLA with minimum amino acid residues. In order to investigate the properties of Cbody-cpBLA, it was

Figure 3. Antigen-dependent catalytic activity of Cbody-cpBLA. The solution of 70 nM Cbody-cpBLA, 1 μg/mL BGP-C10, and 1 μM fluorocillin was mixed in (a) PBS, (b) PBS containing 1 M urea, and (c) PBS containing 0.1% Triton X-100 and 250 μg/mL BSA, incubated at 30 °C, and read for fluorescence. Background fluorescence of the samples without Cbody-cpBLA was subtracted for each condition. Averages of three samples with an error bar of 1 SD are shown. Blue circle indicates the catalytic activity in the presence of 1 μg/mL BGP-C10, and red triangle indicates in the absence of peptide. (d) Dose−response curves for BGP-C10 and BGP-C10dV peptides in the reaction buffer as in (c). The reaction rates at each peptide concentration are shown as the relative value to the rate in the absence of peptides. Statistical analysis was conducted using the two-tailed unpaired Student’s t test: (∗) p < 0.05, (∗∗) p < 0.01. C


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Table 1. Catalytic Parameters Obtained for BLA and Cbody-cpBLA Regarding Nitrocefin Decomposition at 30°C urea fusion protein a

BLA BLA Cbody-cpBLA Cbody-cpBLA a

peptide

(M) 0 1.2 1.2 1.2

(−) (−) 1 μM BGP-C10dV 1 μM BGP-C10

[E]

Vmax

kcat

kcat

Km

kcat/Km

(nM)

(×10−9 M s−1)

(s−1)

relative value

(μM)

(×106 s−1 M−1)

R2

2 70 70

165.8 ± 16.5 3.32 ± 0.62 15.49 ± 2.10

900 82.90 ± 8.27 0.0475 ± 0.0089 0.2212 ± 0.0300

110 414.3 ± 59.7 42.1 ± 28.5 121.4 ± 34.9

8182 200.1 ± 139 1.128 ± 0.311 1.822 ± 0.860

0.999 0.851 0.982

10.9 1 0.00057 0.00267

1 4.66

Reported in ref 27.

fragments is considered important for improving their sensitivity.26,31 At this point, Clampbody is expected to be a useful tool for the creation of a range of antibody-based protein switches.

truncated BGP-C10. Since KTM219 is known to recognize the C-terminus of BGP and cannot bind to BGP-C10dV, this result clearly demonstrates the fine recognition specificity of CbodycpBLA derived from the parental antibody (Figure 3d, SI Figure S3). In order to demonstrate the importance of Fv orientation in the fusion protein, we expressed and purified a fusion protein Fv-cpBLA by connecting the conventional VH and VL to the Nand C-termini of cpBLA, respectively, in the same manner of Cbody-cpBLA. In addition, we prepared another fusion protein (Fv-linker-cpBLA) by connecting VH and VL to cpBLA via (EAAAK)2 helix-prone linkers (SI Figure S4, parts a and b). These fusion proteins were expressed in the inclusion bodies of SHuffle T7 Express lysY cells and prepared in a similar way to Cbody-cpBLA (SI Figure S4, parts c and d). Fv-cpBLA and Fvlinker-cpBLA also showed good antigen-binding activity (SI Figure S5). For analyzing their catalytic activities, the hydrolysis of a chromogenic substrate nitrocefin was investigated. In PBS containing urea, the antigen-dependency of the catalytic activity of Cbody-cpBLA increased as the concentration of urea increased (SI Figures S6, S7a). On the contrary, Fv-cpBLA and Fv-linker-cpBLA showed no dependency to the antigen peptide in any conditions. Therefore, these results clearly indicate the importance of Clampbody format for regulating the stability and catalytic activity of cpBLA (SI Figure S7, parts b and c). As shown in SI Figure S6, the regulation of catalytic activity by Clampbody was most prominent in 1−1.5 M urea. We determined the specific activity kcat and the Michaelis constant Km from the kinetic assays for both Cbody-cpBLA and the wildtype BLA using nonlinear least-squares curve fittings (Table 1, SI Figure S8). In PBS containing 1.2 M urea, the specific activity of Cbody-cpBLA was 0.057% of the wild type BLA.27 However, in the presence of 1 μM BGP-C10, the specific activity significantly increased and the value was 4.66-fold higher than with no antigen peptide (p < 0.01). Therefore, the antigen-dependent catalytic behavior of Cbody-cpBLA was clearly implemented, and the dependency is significantly higher than in the Fv-cpBLA that we reported previously.13 Here we designed a novel antibody variable region format Clampbody that has novel termini near the domain interface and demonstrated that it retains recognition specificity similar to that of the parental antibody. Taking advantage of the proximity of their termini, Clampbody was fused directly to a circularly permutated enzyme to create the molecular switch Cbody-cpBLA, which showed antigen-dependent catalytic activity at suboptimal reaction conditions. Recently, a variety of sensor proteins comprising a ligand-binding protein fused with evolutionally distinct enzymes and protein fragments are reported and used for a number of assays based on PCA,28 fluorescence resonance energy transfer,29 and transduction of reaction intermediate.30 In all of these fusion proteins, the tight control of the distance and orientation of proteins or their

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EXPERIMENTAL SECTION See Supporting Information for more experimental details. Modeling of the Structure of Cbody-cpBLA. The structure of VH and VL of anti-BGP C-terminal peptide antibody cloned from KTM-219 hybridoma was estimated by molecular modeling with Web Antibody Modeling (WAM) located at URL http://antibody.bath.ac.uk.32 The crystal structure of cpBLA of fusion protein RG13 was obtained from RCSB Protein Data Bank (PDB code 4DXB). The structural models were drawn with Pymol (Schrödinger KK, Tokyo, Japan). Construction of the cpVH and cpVL Genes. The DNA fragment encoding cpVH was prepared by splice overlapextension (SOE) PCR of the fragment encoding Gly42 to Ser113 of VH with a linker peptide (G4S)3 at the C-terminus, and that encoding Glu1 to Pro41 with the linker at the Nterminus. Similarly, the DNA fragment encoding cpVL was prepared by SOE PCR of the fragment encoding Gly41 to Arg108 of VL with a (G4S)3 at the C-terminus, and that encoding Asp1 to Pro40 with the linker at the N-terminus. The DNA fragment encoding cpBLA(RG13) was also prepared by SOE PCR of the fragment encoding Trp227 to Trp286 of TEM-1 β-lactamase with a short linker GSGGS at the Cterminus, and that encoding His24 to Gly226 with the linker at the N-terminus. These fragments were inserted to the multiple cloning sites of pET30b(+) (Novagen), resulting in pClampcpBLA. To expression sc-Cbody, the DNA fragment encoding the SG4 linker was inserted into pClamp (pClamp-SG4). Expression and Purification of Proteins. SHuffle T7 Express lysY cells (New England Biolabs Japan, Tokyo, Japan) were transformed with the expression plasmids. For protein expression, single colony was picked and grown overnight at 37 °C in 3 mL of LB medium containing 50 μg/mL kanamycin (LBK), from which 1 mL was used to inoculate 250 mL of LBK medium, The cells were cultured at 37 °C until OD600 reached 0.4, when final 0.4 mM IPTG was added and cultivated overnight at 16 °C. The pelleted cells were homogenized and centrifuged at 10 000g for 60 min at 4 °C. The pellet precipitant was solubilized, and the fusion protein was purified with Talon resin (Clontech, Takara-bio) and refolded as previously described.13 The concentration of purified protein was measured with a Pierce BCA protein assay kit (Thermo scientific). Western Blotting. Proteins were transferred to a nitrocellulose membrane and detected with HRP-conjugated antiHis6 antibody (Roche Diagnostics Japan, Tokyo, Japan). The membrane was washed with TBS (Tris-buffered saline: 50 mM Tris-HCl, 150 mM NaCl, pH7.4) containing 0.05% of Tween D


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Clampbody, a new antibody variable region format that can antigen-dependently clamp protein termini; cp, circularly permutated; ELISA, enzyme-linked immunosorbent assay; Fv, antibody variable region; MBP, maltose binding protein; PBS, phosphate buffered saline; sc, single chain; VH, antibody heavy chain variable region domain; VL, antibody light chain variable region domain

20 and developed with an Amersham ECL Prime (GE Healthcare, U.K.) and a LAS-4000 mini luminoimager (Fujifilm, Tokyo, Japan). ELISA. After streptavidin type II (Wako, Osaka, Japan) was immobilized in Greiner 655061 microplate (Greiner, Tokyo) wells overnight, blocked with 20% Immunoblock (DS Pharma, Osaka, Japan), biotinylated BGP-C11 peptide (biotinQEAYRRFYGPV-COOH) was applied and incubated at 1 h at room temperature. After 3 washes with PBST, samples were applied and incubated at 1 h at room temperature. After 3 washes with PBST, bound protein was probed with HRPconjugated anti-His6 antibody (Roche Diagnostics) and developed with 3,3′5,5′-tetramethylbenzidine (Sigma). BLA Activity Assay. The BLA activity was assayed with the fluorogenic substrate fluorocillin or chromogenic substrate nitrocefin. BGP-C10 (NH2-EAYRRFYGPV-COOH) and BGPC10dV (NH2-EAYRRFYGP-COOH) were used as antigen peptides. For fluorocillin assay, 25 μL of protein in PBS and 25 μL of antigen solution in PBS were mixed and incubated at room temperature for 30 min, and then 50 μL of 2 μM fluorocillin in PBS (containing urea, TritonX-100, Tween-20 or BSA, when specified) was added, incubated at 30 °C for 2 h, and read for their fluorescence intensity in a white microplate (Corning Costar 3693) at 535 nm with 485 nm. Similarly, for nitrocefin assay, 50 μL of mixture of protein and antigen in PBS incubated for 30 min at room temperature was added to 50 μL of 0−500 μM nitrocefin in PBS (containing 0−3 M urea), incubated at 30 °C for 1 h, and read for their absorbance at 486 nm. For both assays, the initial rates of reactions were fit to the Michaelis−Menten equation as follows,

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(1) Abe, R., Ohashi, H., Iijima, I., Ihara, M., Takagi, H., Hohsaka, T., and Ueda, H. (2011) "Quenchbodies": quench-based antibody probes that show antigen-dependent fluorescence. J. Am. Chem. Soc. 133, 17386−17394. (2) Koos, B., Cane, G., Grannas, K., Lof, L., Arngarden, L., Heldin, J., Clausson, C.-M., Klaesson, A., Hirvonen, M. K., de Oliveira, F. M. S., et al. (2015) Proximity-dependent initiation of hybridization chain reaction. Nat. Commun. 6, 7294. (3) Wei, Q., Lee, M., Yu, X., Lee, E. K., Seong, G. H., Choo, J., and Cho, Y. W. (2006) Development of an open sandwich fluoroimmunoassay based on fluorescence resonance energy transfer. Anal. Biochem. 358, 31−37. (4) Ueda, H., Kubota, K., Wang, Y., Tsumoto, K., Mahoney, W., Kumagai, I., and Nagamune, T. (1999) Homogeneous noncompetitive immunoassay based on the energy transfer between fluorolabeled antibody variable domains (open sandwich fluoroimmunoassay). BioTechniques 27, 738−742. (5) Schumacher, F. F., Sanchania, V. A., Tolner, B., Wright, Z. V. F., Ryan, C. P., Smith, M. E. B., Ward, J. M., Caddick, S., Kay, C. W. M., Aeppli, G., et al. (2013) Homogeneous antibody fragment conjugation by disulfide bridging introduces ‘spinostics’. Sci. Rep. 3, 1525. (6) Stains, C. I., Furman, J. L., Porter, J. R., Rajagopal, S., Li, Y., Wyatt, R. T., and Ghosh, I. (2010) A general approach for receptor and antibody-targeted detection of native proteins utilizing splitluciferase reassembly. ACS Chem. Biol. 5, 943−952. (7) Chung, C.-I., Makino, R., Dong, J., and Ueda, H. (2015) Open Flower Fluoroimmunoassay: A general method to make fluorescent protein-based immunosensor probes. Anal. Chem. 87, 3513−3519. (8) Alcalá, P., Ferrer-Miralles, N., and Villaverde, A. (2003) Engineering of Escherichia coli β-galactosidase for solvent display of a functional scFv antibody fragment. FEBS Lett. 533, 115−118. (9) Siemers, N. O., Kerr, D. E., Yarnold, S., Stebbins, M. R., Vrudhula, V. M., Hellstrom, I., Hellstrom, K. E., and Senter, P. D. (1997) Construction, expression, and activities of L49-sFv-betalactamase, a single-chain antibody fusion protein for anticancer prodrug activation. Bioconjugate Chem. 8, 510−519. (10) Afshar, S., Olafsen, T., Wu, A. M., and Morrison, S. L. (2009) Characterization of an engineered human purine nucleoside phosphorylase fused to an anti-her2/neu single chain Fv for use in ADEPT. J. Exp. Clin. Cancer Res. 28, 147. (11) Alderson, R. F., Toki, B. E., Roberge, M., Geng, W., Basler, J., Chin, R., Liu, A., Ueda, R., Hodges, D., Escandon, E., et al. (2006) Characterization of a CC49-Based Single-Chain Fragment−βLactamase Fusion Protein for Antibody-Directed Enzyme Prodrug Therapy (ADEPT). Bioconjugate Chem. 17, 410−418. (12) Yokozeki, T., Ueda, H., Arai, R., Mahoney, W., and Nagamune, T. (2002) A homogeneous noncompetitive immunoassay for the detection of small haptens. Anal. Chem. 74, 2500−2504. (13) Kojima, M., Iwai, H., Dong, J., Lim, S. L., Ito, S., Okumura, K., Ihara, M., and Ueda, H. (2011) Activation of circularly permutated βlactamase tethered to antibody domains by specific small molecules. Bioconjugate Chem. 22, 633−641. (14) Guntas, G., Mitchell, S. F., and Ostermeier, M. (2004) A Molecular Switch Created by In Vitro Recombination of Nonhomologous Genes. Chem. Biol. 11, 1483−1487. (15) Guntas, G., Mansell, T. J., Kim, J. R., and Ostermeier, M. (2005) Directed evolution of protein switches and their application to the creation of ligand-binding proteins. Proc. Natl. Acad. Sci. U. S. A. 102, 11224−11229.

v = Vmax[S]/(K m + [S])

by the nonlinear least-squares algorithm of Kaleidagraph 4.1 (Synergy Software, Reading, PA, USA).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00040. Materials and methods, a table for oligonucleotides used and figures showing the results of analyses of sc-Cbody and Cbody-cpBLA (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Tatsuya Shinoda of Kyowa Medex Co. for generously allowing the use of the genes derived from KTM-219 IgG. This study was supported by Grant-in-Aid for Scientific Research (Grants 24360336 and 15H04191 to H.U. and Grant 46420793 to J.D.) from JSPS, Japan, and partly by Strategic International Collaborative Research Program, Japan Science and technology Agency (JST).

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ABBREVIATIONS USED BGP, bone Gla protein or osteocalcin; BLA, TEM-1 βlactamase; BSA, bovine serum albumin; Cbody, Clampbody; E


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