Covalent and Oriented Surface Immobilization of Antibody Using

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Covalent and Oriented Surface Immobilization of Antibody using Photoactivatable Antibody Fc-Binding Protein Expressed in Escherichia coli Yeolin Lee, Jiyun Jeong, Gabi Lee, Jeong Hee Moon, and Myung Kyu Lee Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02071 • Publication Date (Web): 30 Aug 2016 Downloaded from http://pubs.acs.org on September 2, 2016

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Analytical Chemistry

Figure 1. Residue selection for methionine and arginine mutation of the C3 domain of FcBP. On the basis of the crystal structure (PDB: 1FCC),30 three selected residues (yellow), Q32, N35 and D40, for methionine mutation are in close proximity with the indicated antibody residues (cyan). N37 (yellow) selected for arginine mutation is also indicated. The partial sequence 21-56 of the C3 domain is shown with the mutated residues in the C3PG or 2xC3PG mutant proteins (e.g. 2m: C3PG2m or 2xC3PG2m). 81x82mm (300 x 300 DPI)

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Figure 2. SDS-PAGE analysis of FcBP expression under co-expression of the wild type or mutant methionyl tRNA synthetase (MRS) in the Met auxotroph E. coli cells. The Met-auxotroph E. coli B834 cells were cotransformed with one of pBAD-MRS plasmids and one of pET-FcBP plasmids. MRS and FcBP were sequentially expressed by treatment of 0.02% arabinose in LB broth and 1 mM isopropyl β-D-1thiogalactopyranoside in the M9VC minimal media supplemented with either no additive (-), 50 µg/ml Met or 50 µg/ml pMet as indicated. Total cell lysates were subjected to reducing SDS-PAGE. MRSwt and MRS5m indicate the wild type MRS and the optimized MRS mutant, respectively. The grey and black arrows indicated the expressed MRS and FcBP (PG), respectively. Molecular weights of marker proteins (italicized) are indicated. 64x52mm (300 x 300 DPI)


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Figure 3. Accessibility of the diazirine group of pMet to the binding site of the wild type and MRSs. (A) Complex structure of MRSwt and pMet. The complex structure were built by substitution of pMet (cyan) for Met on the basis of a crystal structure (PDB: 1F4L)33 using a PyMOL program. The numbers indicate the shortest distances (Å) from the diazirine group of pMet to the surround MRSwt residues (pink and yellow) selected for mutagenesis. (B to D) Entrance sizes of wild type and mutant MRSs and accessibility of the side groups of Met or pMet through the entrances. Structures and entrance sizes of wild type and mutant MRSs were predicted by mutagenesis and surface analysis using the PyMOL program. Entrance sizes of MRSwt (blue line), MRS3m (green line) and MRS5m (red line) as well as the mutated residues of MRS5m are indicated (B). The side group of Met passes through all the entrances (C), while the diazirine group of pMet only passes through the MRS5m entrance (D). 81x80mm (299 x 299 DPI)



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Figure 4. Evaluation of photocrosslinking between photoactivatable FcBPs and antibodies by SDS-PAGE. Mixtures of photoactivatable FcBPs and antibodies or human Fc fragments were incubated in the presence (+) or absence (-) UV irradiation and analyzed by SDS-PAGE. SDS-PAGE was performed under reducing conditions unless “nonreducing” indication. (A) Photocrosslinking of photoactivatable 2xC3PG mutant proteins to mammalian serum antibodies derived from human (Hu), rabbit (Rb), goat (Gt) or mouse (Mo). (B) Photocrosslinking of C3PG4m with a C3 domain to EGFR-hmAb (hmAb) or human Fc fragment (hFc). (C) Photocrosslinking of 2xC3PG4m with two C3 domains to hmAb or hFc. Photocrosslinked bands are observed only in the UV-irradiated Ab(Fc)/FcBP mixtures. The indicated protein bands represent reduced heavy chains (H), light chains (L) and hFc fragments (Fc) as well as intact hFc fragments connected by disulfide bonds (Fc*) with or without photocrosslinking to photoactivatable FcBPs (PG). Molecular weights of marker proteins (italicized) are indicated. 56x18mm (300 x 300 DPI)


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Figure 5. Stability of photocrosslinked antibody from serum antibody-induced dissociation. Ni-NTA agarose beads were sequentially incubated with C3PG4m proteins with Met or pMet and biotinylated antibodies (bAbs). The bAb bound beads were either incubated in the presence (+) or absence (-) of UV irradiation, and further incubated either in human sera (+) or 2% BSA-PBS (-). The bead bound proteins eluted by treatment of 150 mM imidazole were subjected to reducing SDS-PAGE. The protein bands on the SDS-PAGE gel were transferred to a nitrocellulose membrane for western blot analysis of the bAb bands. Reduced heavy chains (H) and light chains (L) with photocrosslinked bands (HP) are indicated. 81x58mm (300 x 300 DPI)



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Figure 6. Antigen immunoprecipitation of photocrosslinked antibody in human sera. Photoactivatable C3PG4m-beads were prepared as described in the scheme and incubated with HER2-hmAbs in the presence (+) or absence (-) of UV irradiation. The hmAb beads were further incubated with the HER2 proteins either in human sera (+) or 2% BSA-PBS (-). The bead bound proteins eluted by treatment of 1% SDS were subjected to reducing SDS-PAGE. The protein bands on the gel were either stained with coomassie brilliant blue (CBB) or transferred to a nitrocellulose membrane for western blot (WB) analysis of the HER2 bands. The percent HER2 immunoprecipitation yields are indicated. 81x50mm (300 x 300 DPI)


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Figure 7. Detection of EGFR proteins in human sera using EGFR-hmAb arrays irradiated UV. Antibody arrays were fabricated as described in the scheme. The amounts of hmAbs on the spots were adjusted by controlling the spotted amounts of 2xC3PG4m proteins as described in the Experimental Section. Fluorescence images depending on the EGFR concentrations were monitored by immunostaining using fluorescein-labeled anti-EGFR rat antibody (fαEGFR) at 488 nm excitation and 501-560 nm emission. Fluorescence intensities (A.U.) of the spots were calculated from the fluorescence images.α 82x90mm (300 x 300 DPI)



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Covalent and Oriented Surface Immobilization of Antibody using Photoactivatable Antibody Fc-Binding Protein Expressed in Escherichia coli Yeolin Lee†§, Jiyun Jeong†§, Gabi Lee†, Jeong Hee Moon‡, and Myung Kyu Lee*†§ †

Hazards Monitoring Bionano Research Center, ‡Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, and §Department of Nanobiotechnology, University of Science and Technology, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea ABSTRACT: Fc-specific antibody binding proteins (FcBPs) with the minimal domain of protein G are widely used for immobilization of well-oriented antibodies onto solid surfaces, but the noncovalently bound antibodies to FcBPs are unstable in sera containing large amounts of antibodies. Here we report novel photoactivatable FcBPs with photomethionine (pMet) expressed in E. coli, which induce Fc-specific photocrosslinking with antibodies upon UV irradiation. Unfortunately, pMet did not support protein expression in the native E. coli system, and therefore we also developed an engineered methionyl tRNA synthetase (MRS5m). Co-expression of MRS5m proteins successfully induced photoactivatable FcBP overexpression in methionine-auxotroph E. coli cells. The photoactivatable FcBPs could be easily immobilized on beads and slides via their N-terminal cysteine residues and 6xHis tag. The antibodies photocrosslinked onto the photoactivatable FcBP-beads were resistant from serum-antibody mediated dissociation and efficiently captured antigens in human sera. Furthermore, photocrosslinked antibody arrays prepared using this system allowed sensitive detection of antigens in human sera by sandwich immunoassay. The photoactivatable FcBPs will be widely applicable for well-oriented antibody immobilization on various surfaces of microfluidic chips, glass slides and nanobeads, which are required for development of sensitive immunosensors. Stable and efficient immobilization of antibodies (Abs) with well-exposed orientation of antigen binding sites on solid surfaces is a key step in development of highly sensitive immunosensors for diagnosis of various pathogens and diseases.1,2 Covalent conjugation of Abs onto solid surfaces using chemical linkers often results in significant reduction of antigen binding affinity to the immobilized Abs due to random orientation of Abs as well as modification of their antigen binding sites.3,4 To overcome these problems, a variety of methods has been developed to achieve site-specific Ab immobilization onto solid surfaces.2 Abs can be immobilized onto the surfaces via their carbohydrate moieties activated to aminereactive aldehyde groups, but the need for exquisite control and optimization of Ab oxidation are inevitable.5,6 Abs are also conjugated onto the surfaces via free sulfhydryl groups exposed by site-specific reduction of disulfide bonds between the heavy chains.6,7 However, the strategy is hard to be generalized against various Abs with several inter- and intra-molecular disulfide bonds. Protein A and G have been widely used for Fc-specific Ab immobilization providing wellexposed orientation of antigen binding sites on solid surfaces.1,3 Many strategies for Fcspecific Ab immobilization onto the surfaces have been developed using the Fc binding proteins.8-10 However, the noncovalently bound Abs are reversibly dissociated from the Fc binding proteins during subsequent steps.11 To overcome this limitation, the maleimidefunctionalized benzophenone molecule was chemically introduced into the cysteine (Cys) mutated minimal Fc binding domain of protein G.12 However, this approach prevents to use the Cys residue for the protein immobilization on the surfaces.



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Recent techniques allow site-specific incorporation of noncanonical amino acids (NAAs) into proteins during in vivo translation in Escherichia coli (E. coli). Although some NAAs can be incorporated into recombinant proteins using the native translational system,13,14 but mutations of amino-acyl tRNA synthetases (aaRSs) are needed for efficient NAA incorporation.15,16 Genetic code expansions using engineered orthogonal aaRS/tRNA pairs in response to the UAG-amber-stop codon allow site-selective incorporations of NAAs into proteins by E. coli, yeast, and mammary cells.17-19 The systems have also proven effective for expression of proteins including photoactivatable amino acids with diazirine,20-22 benzophenone,23-25 and azido groups26 in E. coli. Recently, the NAA with a benzophenone group, benzoyl-phenylalanine (BPA), has been introduced into the mutated protein Z derived from the B domain of protein A using the engineered in vivo translational system,27 and the photoactivatable protein Z mutants with BPA have successfully induced the photocrosslinking with Abs.24,25 In this study, we aimed to develop novel photoactivatable Fc-specific antibody binding proteins (FcBPs) with photomethionine (pMet) to induce Fc-specific photocrosslinking with Abs. pMet with unique similarity to methionine (Met) is readily activated by the less harmful 365 nm UV for photocrosslinking. Although pMet was successfully incorporated into target proteins using a native mammalian translation system,28 we found that Met-auxotroph E. coli cells did not allow significant expression of recombinant proteins in pMet-supplemented minimal media. The problem was solved by mutation of E. coli methionyl tRNA synthetase (MRS) to fit in well with pMet. Here we report successful overexpression of photoactivatable FcBPs with pMet under co-expression of the optimized MRS mutant in Met-auxotroph E. coli cells. Design, construction and expression of the mutant FcBPs and MRSs are described. Fc specific-photocrosslinking between Abs and photoactivatable FcBPs were evaluated. We further investigated advantages and applications of photoactivatable FcBPs for covalent immobilization of Abs onto various surfaces and immunodiagnosis of antigens in human sera. EXPERIMENTAL SECTION Materials. Met and pMet (L-2-amino-5,5-azi-hexanoic acid) were purchased from SigmaAldrich and Thermo Scientific, respectively. The mixture of 19 amino acids without Met (CSM-Met) was obtained from MP Biomedicals. Bovine serum albumin (BSA), human sera, horse-radish peroxidase (HRP)-labeled streptavidin (streptavidin-HRP) and various purified serum Abs were purchased from Sigma-Aldrich. Human epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) with a C-terminal 6xHis group were purchased from Sino biological Inc. (China). Human Fc (hFc) fragment was purchased from Abcam. Humanized monoclonal Abs, EGFR-hmAb (cetuximab) and HER2hmAb (trastuzumab), were obtained from Merck Serono and Roche, respectively. Ni-NTAagarose beads and Sulfolink-agarose beads were purchased for Qiagen and Thermo Scientific, respectively. Restriction enzymes, BamHI, ClaI, NcoI, KpnI, XbaI and XhoI, were purchased from New England Biolabs. Construction of Plasmids Encoding Mutant FcBPs. The primer DNAs used for construction of plasmids encoding mutant FcBPs are summarized in Table S1 in the Supporting Information. The DNA sequence encoding three Met residues between the NdeI and BamHI sites of the pET-28a(+) vector (Novagen, EMD Millipore) was initially deleted by the PCR method (for details, see Figure S1 in the Supporting Information). The resulting pET-28at vector was used for construction of the plasmids encoding FcBPs. A DNA encoding a 2xC3PGwt protein with two C3 domains was amplified by PCR using the primers, PG1 and PG2, against pET-2xFcBD,12 and cloned into the BamHI/XhoI sites of the pET-28at vector as shown in Figure S2 in the Supporting Information. The resulting pET-


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2xC3PGwt plasmid was further mutated to construct the pET-2xC3PG mutant plasmids (For details, see Figure S3 in the Supporting Information). The pET-C3PG mutant plasmids were constructed using the corresponding pET-2xC3PG mutant plasmids (For details, see Figure S4 in the Supporting Information). Construction of Plasmids for Mutant MRS Proteins. Primer DNAs used for construction of wild type and mutant MRS plasmids are summarized in Table S2 in the Supporting Information. The detail scheme and method are described in Figure S5 in the Supporting information. Briefly, a DNA encoding the C-terminal truncated MRS sequence (1-548) was amplified by PCR using the primers, PM1 and PM2, against the E. coli BL21 genomic DNA, and cloned into the NcoI/KpnI sites of the pBAD/Myc-HisA vector (Invitrogen). The resulting pBAD-MRSwt plasmid encoding a truncated wild type MRS, was further mutated to construct the pBAD-MRS mutant plasmids as described in Figure S5 in the Supporting Information. Expression of Photoactivatable FcBPs. Met auxotroph E. coli B834 cells were transformed with one of the plasmids encoding FcBPs and cultured in LB broth with kanamycin until absorbance 1.0-1.2 at 620 nm. The cells were washed with M9 buffer (48 mM Na2HPO4, 22 mM KH2PO4, 9 mM NaCl, and 19 mM NH4Cl), and then starved in 100 ml M9VC media (see Supporting Information) for 2 h at 37 °C. The starved cells were divided into 3 parts, and each part was adjusted to 100 ml of M9VC media supplemented with either no additive, 50 µg/ml Met or 50 µg/ml pMet. FcBP expression was induced by treatment of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 16 to 18 h at 20 °C, and subjected to reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). To investigate the effect of exogenous MRS proteins on expression of photoactivatable FcBPs, one of pBAD-MRS plasmids and one of mutated FcBP plasmids were co-transformed into Met-auxotroph E. coli cells. The co-transformed cells were cultured in LB broth with ampicillin and kanamycin until absorbance 0.8-0.9 at 620 nm, and exogenous MRS expression was induced by treatment of 0.02% L-arabinose for 2 h at 37 °C. The cells were washed with the M9 buffer, and then starved in 100 ml M9VC media for 2 h at 37 °C. The remaining steps were followed the above procedures. Purification and Mass Analysis of FcBPs. FcBPs were purified by a Ni-NTA-agarose chromatography (for details, see Figure S6 in Supporting Information). The purified FcBPs were subjected to reducing SDS-PAGE and confirmed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) using a Q-TOF mass spectrometer (Waters, England) after in-gel trypsin digestion as described previously.29 To determine pMet-incorporation into a whole photoactivatable FcBP, the purified C3PG3m proteins expressed in either Met- or pMet-supplemented media were desalted using a C18 column and analyzed by electrospray ionization mass spectrometry (ESI-MS) using the Q-TOF mass spectrometer. Photocrosslinking Analysis of Photoactivatable FcBPs to Abs. For photocrosslinking, following incubation of various Abs or human Fc (hFc) fragments with FcBPs for 30 min at room temperature, the mixtures were irradiated with a hand held 365 nm UV lamp on ice for 30 min. The UV-irradiated mixtures were subjected to reducing or nonreducing SDS-PAGE, and the band intensities were measured by densitometric analysis using an ImageJ program (NIH, USA). Serum Stability of Photocrosslinked Abs. The mixture of 5 mg EGFR-hmAb and 10fold molar excess biotin-hydroxysuccinimide (NHS) was incubated for 2 h to prepare biotinylated Ab (bAb), and the unreacted biotin-NHS was removed by dialysis. To prepare cleavable FcBP-beads, 10 µg C3PG4m with Met or pMet in TBS (20 mM Tris-HCl and 150



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mM sodium chloride, pH 7.5) was incubated with 10 µl Ni-NTA-agarose beads for 30 min. After washing the beads with TBST (0.05% tween 20 in TBS), 25 µg bAb in TBS was incubated with the C3PG4m bound beads for 30 min on ice in the presence or absence of UV irradiation. The bAb-bound beads were washed with TBST and incubated in 100 µl of human sera or 2% bovine serum albumin in PBS (BSA-PBS) for 1 h. After removing the unbound proteins with TBST washing, the bead bound proteins were eluted with 150 mM imidazole in TBS. The eluted proteins were subjected to reducing SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad) for western blot (WB) analysis. The membrane was incubated with streptavidin-HRP in 5% nonfat milk-TBS for 1 h, and the bAbs were monitored by chemiluminescence using Amersham ECL western blotting detection reagents (GE healthcare). Antigen Immunoprecipitation using Photocrosslinked Ab-Beads. Ten µg of photoactivatable C3PG4m was immobilized on 10 µl of sulfolink-agarose beads through their N-terminal cysteine residues. The modified beads were incubated with 25 µg HER2-hmAb for 30 min on ice in the presence or absence UV irradiation. After washing the beads with TBST, the hmAb-bound beads were incubated with 5 µg of recombinant HER2 proteins with a C-terminal 6xHis tag spiked in 100 µl of human sera or 2% BSA-PBS for 1 h. Excess unbound proteins were washed with TBST, the bound proteins were eluted with 1% SDS in PBS. The eluted proteins were subjected to reducing SDS-PAGE and transferred onto a nitrocellulose membrane for WB analysis. The membrane was sequentially incubated with anti-6xHis mouse mAb and goat anti-mouse IgG-HRP (Millipore) in 5%-nonfat milk-TBS, and the HER2 proteins were monitored by chemiluminescence. Detection of Antigens in Human Sera Using Ab Array. An aldehyde-activated glass slide (Arrayit Co.) was sequentially incubated in 100 mM 1,2-Bis(2-aminoethoxy)ethane, pH 7.5, for 1 h and 1% sodium cyanoborohydride for 1 h. The amine activated slide was further incubated in 1 mg/ml NHS-3-maleimidopropionate in PBS. The serially diluted photoactivatable FcBPs in PBS were applied onto the maleimide activated slide using a 300 µm mask (Arrayit Co.). The free maleimide groups were deactivated by treatment of 10 mM 2-mercaptoethanol in 2% BSA-TBS, and the FcBP-modified slide was then incubated with 40 µg/ml EGFR-hmAbs in 2% BSA-TBS in the presence or absence of 365 nm UV irradiation for 30 min on ice. The Ab array slide was incubated in the premixed solution with the serially diluted EGFR and fluorescein-labeled anti-EGFR rat mAb (Abcam) in human sera for 1 h. The slide was washed with TBST, briefly cleaned with water, and dried by nitrogen blowing. Fluorescence images on the slide were monitored using a GenePix 4200 microarray scanner (Axon Instruments Inc.) using a 488 nm excitation laser and a 510-560 emission filter. RESULTS AND DISCUSSION Design and Mutagenesis of Photoactivatable FcBPs. We aimed covalent immobilization of Abs with well-exposed orientation using photoactivatable FcBPs with pMet on various surfaces. Since pMet uses the AUG codon,28 we initially constructed a pET-28at vector with removing a sequence containing 3 AUG codons between NdeI and BamHI sites of the pET28a(+) vector (Figure S1 in the Supporting Information). The FcBPs with one or two C3 domains of protein G (C3PG or 2xC3PG, respectively) were designed to have additional two consecutive Cys residues and a 6xHis tag at their N-terminus (Figures S2 to S4 in the Supporting Information). On the basis of a complex crystal structure (PDB: 1FCC),30 three residues in the C3 domain, Q32, N35 and D40, were selected for Met mutation. The residues are in close proximity enough for direct contact with some Ab residues indicated (Figure 1).


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The N37 residue of FcBP is important for Fab binding,31 and the N37Y mutation abolishes the binding.12 We mutated the residue to arginine instead of tyrosine to offer a hydrophilic and bulk side group (Figure 1). Effect of Mutant MRS on Photoactivatable FcBP Expression. FcBP overexpression in Met auxotroph E. coli cells was failed in pMet-supplemented media, but was successful in Met-supplemented media (data not shown). Although exogenous MRSwt co-expression helps recombinant protein expression with some Met analogs in Met auxotroph E. coli cells,13,32 MRSwt co-expression did not allow FcBP expression in pMet-supplemented media (Figure 2A). We hypothesized that the Met binding site of MRSwt may be inappropriate for pMet with a diazirine group. On the basis of the Met-binding MRSwt crystal structure (PDB: 1F4L),33 we built a pMetbinding MRSwt structure using a PyMOL program (Schrödinger, Inc.). Five amino acid residues were identified to be in close proximity with the diazirine moiety of pMet (Figure 3A). Although the L13, Y260 and H301 mutations successfully was reported to allow efficient incorporations of some NAAs into recombinant proteins,34,35 co-expression of MRS3m with L13G, Y260F and H301L mutations also failed FcBP overexpression in pMetsupplemented media. The predicted entrance size of MRS3m is similar to MRSwt (Figure 3B). The entrance sizes of MRSwt and MRS3m are suitable for access of the Met side group (Figure 3C), but are narrow to pass through the diazirine group of pMet (Figure 3D). The additional A12G and I297V mutations were predicted to offer an enlarged entrance suitable for access of the diazirine group (Figures 3B and 3D). Co-expression of MRS5m with A12G, L13S, Y260F, I297V and H301L mutations indeed induced FcBP overexpression in pMetsupplemented media (Figure 2B). These suggest that the A12 and I297 residues of MRSwt act as a primary barrier to pMet access and MRS5m actively catalyzes pMet-tRNA conjugation required for pMet incorporation. To the best of our knowledge, this is the first report describing the A12 and I297 mutations of MRS to introduce NAA. The L13S, Y260F and H301L mutations are considered to provide an enlarged space for the diazirine binding. Expression yields of photoactivatable FcBPs were 6 to 8 mg per 100 ml culture under MRS5m co-expression. Mass Analysis of Purified FcBPs. Three to 5 mg of photoactivatable FcBPs were purified by Ni-NTA chromatography per 100 ml culture. Interestingly, the purified 2xC3PG2m proteins expressed in either Met- or pMet-supplemented media were observed to form SDS-resistant dimeric and/or oligomeric bands on the reducing SDS-PAGE gel (Figure S6 in the Supporting Information). Formation of SDS-resistant dimeric and/or oligomeric bands is considered to be related to increase of hydrophobicity by Met mutation and/or introduction of two Cys residues at the 2xC3PG4m N-terminus. In fact, increase of hydrophilicity by the N37R mutation of 2xC3PG3m revealed significant reduction of the dimer/oligomer formation (Figure S6 in the Supporting Information). The whole mass (8425.8 Da) of C3PG3m protein with Met is agreed to the calculated one with N-terminal Met deletion (8427.3 Da) (Figure S7 in the Supporting information). In fact, the N-terminal Met deletion is frequently occurred by endogenous Met aminopeptidase in E. coli.36 The purified C3PG3m expressed in pMet-supplemented media showed three consecutive mass peaks with approximate 8 Da differences, and the first peak was overlapped with that with Met. Since pMet has an 8 Da higher mass than Met and a C3PG3m molecule contains two internal Met sites, the first, second and third peaks are expected to contain Met/Met (15%), pMet/Met (25%) and pMet/pMet (60%) combinations, respectively. Therefore, further improvement of pMet incorporation into the FcBPs is needed. Photocrosslinking of Abs to Photoactivatable FcBPs. Figure 4A shows photocrosslinking efficiencies of mammalian serum Abs to photoactivatable FcBPs. The Abs



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were incubated with 10-fold molar excess FcBPs to obtain a fully saturated level of photocrosslinking. The FcBPs with Met were not photocrosslinked to the Abs upon 1 h UV irradiation (data not shown), while the photoactivatable FcBPs with pMet were efficiently photocrosslinked only to Ab heavy chains upon 30 min UV irradiation with different efficiencies, under reducing conditions. The photoactivatable 2xC3PG4m proteins with 3 internal Met sites induced approximate 50% photocrosslinking with human and rabbit Abs and 42% photocrosslinking with goat Abs (4m, UV+), while the other photoactivatable FcBPs with 2 internal Met sites induced 30 to 38% photocrosslinking with the Abs (2m and 3m, UV+). These results suggest that the introduced pMet residues in FcBPs enable UVinduced photocrosslinking with the Ab heavy chains. However, the photoactivatable FcBPs revealed less photocrosslinking efficiency to mouse Abs. The mutated Met residues locate in close proximity to the C-terminal heavy chain sequence, LHNHYTQKS, especially with the underlined residues (Figure 1). The sequence is preserved in a variety of mammalian Abs derived from human, rabbit, cow, pig, goat, monkey and so on, while is significantly different from mouse Abs, LHNHHTEKS (IgG1), LHNHHTTKS (IgG2a), LKNYYLKKS (IgG2b) and LHNHHTQKN (IgG3). Taken together, the conserved Ab sequence may be responsible for Ab photocrosslinking ability to the photoactivatable FcBPs. Photoactivatable C3PG4m proteins with a C3 domain were photocrosslinked to approximate 50% of hmAbs and 48% of hFc fragments under reducing conditions (Figure 4B, lanes 2 and 4, respectively). An intact Ab molecule can be photocrosslinked with up to two FcBPs, and this can be analyzed by nonreducing SDS-PAGE. We used hFc fragments (intact MW: 55KD) instead of hmAbs (intact MW: 160 KD) for fine analysis. The nonreducing gel showed that approximate 70% of intact hFc fragments (Fc*) were photocrosslinked with either one or two C3PG4m proteins (45% Fc*P or 25% Fc*2P, respectively) (Figure 4B, lane 6). The photocrosslinking yield for intact Abs can be theoretically estimated using the percent ratio of free heavy chain obtained from reducing conditions (%fHr) as following, [1(%fHr/100)2]x100 (%). Since %fHr for the hmAbs is 50%, 75% of intact hmAbs are estimated to be photocrosslinked to one or two C3PG4m proteins. A photoactivatable 2xC3PG4m molecule with two C3 domains can be photocrosslinked with up to two Abs. In fact, the reducing SDS-PAGE gel showed two different photocrosslinked bands (HP and 2HP) in the UV-irradiated mixture of the hmAbs and the photoactivatable 2xC3PG4m proteins (Figure 4C, lane 2). The similar SDS-PAGE patterns were also observed using the hFc fragments (Figure 4C, lane 4). The results indicate that the photoactivatable FcBP with two C3 domains is more efficient to immobilize Abs on solid surfaces than that with a C3 domain. Stability and Antigen Capture Ability of Photocrosslinked Abs in Human Sera. To discriminate the target Abs from serum Abs, EGFR-hmAbs were initially biotinylated by incubation with 10-fold molar excess biotin-NHS for 1 h. Interestingly, the amine-reactive hmAb biotinylation did not significantly change its photocrosslinking ability, while dramatically reduced its antigen binding affinity (data not shown). Ni-NTA agarose beads were used for capture of FcBPs to allow selective elution of FcBP binding proteins using 150 mM imidazole. The modified beads were incubated with bAbs in the presence or absence of UV irradiation, followed by incubation in human sera or 2% BSA-PBS. The bead bound proteins eluted by 150 imidazole contained FcBPs and Abs (Figure S8 in the Supporting Information). Figure 5 shows WB patterns of the recovered bAbs depending on treatment of UV and/or human sera. Interestingly, the hmAb light chains were more intensively biotinylated than the heavy chains. Since the light chain is not modified by photocrosslinking, the light chain intensity reflects total recovered bAbs. Regardless of UV irradiation, human sera induced approximate 80 % dissociation of bAbs from the FcBPs with Met (lanes 2 and 4


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vs lanes 1 and 3, respectively). Moreover, human sera induced dissociation of most noncovalently bound bAbs from photoactivatable FcBPs with pMet (lane 5), indicating that the diazirine group of pMet is more susceptible to serum Ab-mediated dissociation that that of Met. In contrast, the dissociation was almost completely protected by UV irradiation (lane 8 vs lane 7), demonstrating that the photocrosslinked Abs to FcBPs are stable in human sera. Antigen capture ability of photocrosslinked Abs in human sera was investigated by the immunoprecipitation method. The photocrosslinked HER2-hmAb beads were prepared as shown in the scheme of Figure 6. The Ab-modified beads were incubated with HER2 proteins with a C-terminal 6xHis tag spiked in human sera or 2% BSA-PBS. The HER2 proteins could be sensitively monitored by WB analysis using anti-6xHis Abs. UV irradiation induced 3times more HER2 immunoprecipitation than no UV irradiation even in the absence of human sera (Figure 6, 25% vs 8%). Since the antigen-Ab binding is not affected by 365 nm UV irradiation,12 less HER2 recovery in the absence of UV irradiation is easily inferred to be caused by spontaneous dissociation of noncovalently bound hmAbs from the photoactivatable FcBPs during the subsequent steps. Moreover, less than 2% of HER2 proteins in human sera were immunoprecipitated using no UV irradiated hmAb-beads, also indicating the serum Abmediated hmAb dissociation as shown in Figure 5. In contrast, the HER2 proteins in human sera were rather efficiently immunoprecipitated using UV-irradiated hmAb-beads than those in 2% BSA-PBS (35% vs 25%). Although we cannot explain the unexpected result, the photocrosslinked hmAbs efficiently capture HER2 proteins in human sera. Detection of Antigens in Human Sera using Photocrosslinked Ab Arrays. To demonstrate the utility of the photocrosslinked Abs for detection of antigens in human sera, the EGFR-hmAb array was fabricated as shown the scheme in Figure 7. The 2xC3PG4m proteins were much more efficient to immobilize the hmAbs than the C3PG4m ones on the glass surface (data not shown). The spotted Ab amounts on the array were controlled by spotting the serially diluted photoactivatable FcBPs on the maleimide-activated glass slide. The EGFR proteins in human sera were measured in a single step by direct incubation of the mixture of serially diluted EGFR proteins and fluorescein-labeled anti-EGFR Abs in human sera. No UV-irradiated Ab array was not effective for detection of 18 nM EGFR proteins in human sera unlike UV-irradiated one (Figure S9 in the Supporting Information). There was a clear trend of dose-dependent antigen bindings on UV-irradiated hmAb arrays, and the saturated Ab coverages were observed at above 110 fmol/spot of photoactivatable FcBPs (Figure 7). The results demonstrate that the photoactivatable FcBPs is useful for development of sensitive immunoassay systems by providing covalent and well-oriented Ab immobilization on various surfaces, which is stable in human sera. Recently, Ab arrays have been developed using a photoactivatable bifunctional chemical probe with a boronic acid and a diazirine group, and proved to be effective for sensitive antigen detection.37 However, the detection was performed only in BSA solution.

CONCLUSIONS We have developed a novel system to produce photoactivatable FcBPs with pMet in Met auxotroph E. coli cells. The strategy involved the MRS mutation to express recombinant proteins with pMet and the FcBP mutation to introduce pMet. The optimized MRS5m allowed overexpression of photoactivatable FcBPs with pMet. The optimized photoactivatable FcBPs efficiently induced Fc-specific photocrosslinking with Abs upon UV irradiation. The photoactivatable FcBPs could be site-selectively immobilized on the NiNTA- or maleimide-activated surfaces through their N-terminal 6xHis motif or Cys residues, respectively. The photocrosslinked Ab-beads were stable and enabled efficient capture of



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antigens in human sera. The Ab arrays prepared using this system also allowed sensitive detection of antigens in human sera. Currently, functional nanobeads such as fluorescent nanobeads, quantum dots and gold nanoparticles have been widely used for conjugation of detection Abs required for sensitive immunodiagnosis.38,39 Since the present system is effective for well-oriented Ab immobilization on the bead surfaces as shown in Figures 5 and 6, it will be also applicable for fabrication of well-oriented detection Abs on nanobeads. In this respect, Ab immobilization via the photoactivatable FcBPs will provide a simple and versatile platform for fabricating Ab chips, arrays and nanobeads, which are required for development of sensitive immunosensors. ASSOCIATED CONTENT

Supporting Information Composition of M9VC minimal media; PCR DNA primers for construction of plasmids encoding mutated FcBPs (Table S1) and mutated MRS proteins (Table S2); Construction schemes of the pET-28at vector (Figure S1), the pET-2xC3PGwt plasmid (Figure S2), the pET-2xC3PG mutant plasmids (Figure S3), the pET-C3PG mutant plasmids (Figure S4) and pBAD-MRS mutant plasmids (Figure S5); Purification of FcBPs and SDS-PAGE profiles (Figure S6); ESI-MS mass profiles of purified FcBPs (Figure S7); SDS-PAGE profiles of FcBP-bound proteins (Figure S8); Comparison of detection sensitivity of EGFR proteins in human sera using EGFR-hmAb arrays depending on UV irradiation (Figure S9). AUTHOR INFORMATION Corresponding author *E-mail: [email protected]. Tel: +82 42 8604169 Fax: 82 42 8794593. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the Converging Research Center Program (2014048792) and the 2014 Daedoek Innopolis project, which are funded by the Ministry of Science, ICT and Future Planning; and KRIBB initiative program of Republic of Korea.

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(8) Seo, J. S.; Lee, S.; Poulter, C. D. J Am Chem Soc 2013, 135, 8973-8980. (9) Jung, Y.; Lee, J. M.; Jung, H.; Chung, B. H. Anal Chem 2007, 79, 6534-6541. (10) Lee, J. M.; Park, H. K.; Jung, Y.; Kim, J. K.; Jung, S. O.; Chung, B. H. Anal Chem 2007, 79, 2680-2687. (11) Saleemuddin, M. Adv Biochem Eng Biotechnol 1999, 64, 203-226. (12) Jung, Y.; Lee, J. M.; Kim, J. W.; Yoon, J.; Cho, H.; Chung, B. H. Anal Chem 2009, 81, 936-942. (13) Kiick, K. L.; Weberskirch, R.; Tirrell, D. A. FEBS Lett 2001, 502, 25-30. (14) Link, A. J.; Vink, M. K.; Tirrell, D. A. J Am Chem Soc 2004, 126, 10598-10602. (15) Datta, D.; Wang, P.; Carrico, I. S.; Mayo, S. L.; Tirrell, D. A. J Am Chem Soc 2002, 124, 5652-5653. (16) Kirshenbaum, K.; Carrico, I. S.; Tirrell, D. A. Chembiochem 2002, 3, 235-237. (17) Wang, Y. S.; Fang, X.; Chen, H. Y.; Wu, B.; Wang, Z. U.; Hilty, C.; Liu, W. R. ACS Chem Biol 2013, 8, 405-415. (18) Davis, L.; Chin, J. W. Nat Rev Mol Cell Biol 2012, 13, 168-182. (19) Xie, J.; Schultz, P. G. Nat Rev Mol Cell Biol 2006, 7, 775-782. (20) Tippmann, E. M.; Liu, W.; Summerer, D.; Mack, A. V.; Schultz, P. G. Chembiochem 2007, 8, 2210-2214. (21) Zhang, M.; Lin, S.; Song, X.; Liu, J.; Fu, Y.; Ge, X.; Fu, X.; Chang, Z.; Chen, P. R. Nat Chem Biol 2011, 7, 671-677. (22) Chou, C.; Uprety, R.; Davis, L.; Chin, J. W.; Deiters, A. Chemical Science 2011, 2, 480483. (23) Chin, J. W.; Martin, A. B.; King, D. S.; Wang, L.; Schultz, P. G. Proc Natl Acad Sci U S A 2002, 99, 11020-11024. (24) Hui, J. Z.; Al Zaki, A.; Cheng, Z.; Popik, V.; Zhang, H.; Luning Prak, E. T.; Tsourkas, A. Small 2014, 10, 3354-3363. (25) Yu, F.; Jarver, P.; Nygren, P. A. PloS one 2013, 8, e56597. (26) Chin, J. W.; Santoro, S. W.; Martin, A. B.; King, D. S.; Wang, L.; Schultz, P. G. J Am Chem Soc 2002, 124, 9026-9027. (27) Young, T. S.; Ahmad, I.; Yin, J. A.; Schultz, P. G. Journal of Molecular Biology 2010, 395, 361-374. (28) Suchanek, M.; Radzikowska, A.; Thiele, C. Nat Methods 2005, 2, 261-267. (29) Wilm, M.; Shevchenko, A.; Houthaeve, T.; Breit, S.; Schweigerer, L.; Fotsis, T.; Mann, M. Nature 1996, 379, 466-469. (30) Sauer-Eriksson, A. E.; Kleywegt, G. J.; Uhlen, M.; Jones, T. A. Structure 1995, 3, 265278. (31) Derrick, J. P.; Wigley, D. B. Journal of Molecular Biology 1994, 243, 906-918. (32) Kiick, K. L.; Tirrell, D. A. Tetrahedron 2000, 56, 9487-9493. (33) Serre, L.; Verdon, G.; Choinowski, T.; Hervouet, N.; Risler, J. L.; Zelwer, C. Journal of Molecular Biology 2001, 306, 863-876. (34) Tanrikulu, I. C.; Schmitt, E.; Mechulam, Y.; Goddard, W. A., 3rd; Tirrell, D. A. Proc Natl Acad Sci U S A 2009, 106, 15285-15290. (35) Yoo, T. H.; Tirrell, D. A. Angew Chem Int Ed Engl 2007, 46, 5340-5343. (36) Hirel, P. H.; Schmitter, M. J.; Dessen, P.; Fayat, G.; Blanquet, S. Proc Natl Acad Sci U S A 1989, 86, 8247-8251. (37) Adak, A. K.; Li, B. Y.; Huang, L. D.; Lin, T. W.; Chang, T. C.; Hwang, K. C.; Lin, C. C. ACS Applied Materials & Interfaces 2014, 6, 10452-10460. (38) Djoba Siawaya, J. F.; Roberts, T.; Babb, C.; Black, G.; Golakai, H. J.; Stanley, K.; Bapela, N. B.; Hoal, E.; Parida, S.; van Helden, P.; Walzl, G. PloS One 2008, 3, e2535.



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(39) Deerinck, T. J. Toxicologic Pathology 2008, 36, 112-116. Figure legends Figure 1. Residue selection for methionine and arginine mutation of the C3 domain of FcBP. On the basis of the crystal structure (PDB: 1FCC),30 three selected residues (yellow), Q32, N35 and D40, for methionine mutation are in close proximity with the indicated antibody residues (cyan). N37 (yellow) selected for arginine mutation is also indicated. The partial sequence 21-56 of the C3 domain is shown with the mutated residues in the C3PG or 2xC3PG mutant proteins (e.g. 2m: C3PG2m or 2xC3PG2m). Figure 2. SDS-PAGE analysis of FcBP expression under co-expression of the wild type or mutant methionyl tRNA synthetase (MRS) in the Met auxotroph E. coli cells. The Metauxotroph E. coli B834 cells were co-transformed with one of pBAD-MRS plasmids and one of pET-FcBP plasmids. MRS and FcBP were sequentially expressed by treatment of 0.02% arabinose in LB broth and 1 mM isopropyl β-D-1-thiogalactopyranoside in the M9VC minimal media supplemented with either no additive (-), 50 µg/ml Met or 50 µg/ml pMet as indicated. Total cell lysates were subjected to reducing SDS-PAGE. MRSwt and MRS5m indicate the wild type MRS and the optimized MRS mutant, respectively. The grey and black arrows indicated the expressed MRS and FcBP (PG), respectively. Molecular weights of marker proteins (italicized) are indicated. Figure 3. Accessibility of the diazirine group of pMet to the binding site of the wild type and MRSs. (A) Complex structure of MRSwt and pMet. The complex structure were built by substitution of pMet (cyan) for Met on the basis of a crystal structure (PDB: 1F4L)33 using a PyMOL program. The numbers indicate the shortest distances (Å) from the diazirine group of pMet to the surround MRSwt residues (pink and yellow) selected for mutagenesis. (B to D) Entrance sizes of wild type and mutant MRSs and accessibility of the side groups of Met or pMet through the entrances. Structures and entrance sizes of wild type and mutant MRSs were predicted by mutagenesis and surface analysis using the PyMOL program. Entrance sizes of MRSwt (blue line), MRS3m (green line) and MRS5m (red line) as well as the mutated residues of MRS5m are indicated (B). The side group of Met passes through all the entrances (C), while the diazirine group of pMet only passes through the MRS5m entrance (D). Figure 4. Evaluation of photocrosslinking between photoactivatable FcBPs and antibodies by SDS-PAGE. Mixtures of photoactivatable FcBPs and antibodies or human Fc fragments were incubated in the presence (+) or absence (-) UV irradiation and analyzed by SDS-PAGE. SDS-PAGE was performed under reducing conditions unless “nonreducing” indication. (A) Photocrosslinking of photoactivatable 2xC3PG mutant proteins to mammalian serum antibodies derived from human (Hu), rabbit (Rb), goat (Gt) or mouse (Mo). (B) Photocrosslinking of C3PG4m with a C3 domain to EGFR-hmAb (hmAb) or human Fc fragment (hFc). (C) Photocrosslinking of 2xC3PG4m with two C3 domains to hmAb or hFc. Photocrosslinked bands are observed only in the UV-irradiated Ab(Fc)/FcBP mixtures. The indicated protein bands represent reduced heavy chains (H), light chains (L) and hFc fragments (Fc) as well as intact hFc fragments connected by disulfide bonds (Fc*) with or without photocrosslinking to photoactivatable FcBPs (PG). Molecular weights of marker


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proteins (italicized) are indicated. Figure 5. Stability of photocrosslinked antibody from serum antibody-induced dissociation. Ni-NTA agarose beads were sequentially incubated with C3PG4m proteins with Met or pMet and biotinylated antibodies (bAbs). The bAb bound beads were either incubated in the presence (+) or absence (-) of UV irradiation, and further incubated either in human sera (+) or 2% BSA-PBS (-). The bead bound proteins eluted by treatment of 150 mM imidazole were subjected to reducing SDS-PAGE. The protein bands on the SDS-PAGE gel were transferred to a nitrocellulose membrane for western blot analysis of the bAb bands. Reduced heavy chains (H) and light chains (L) with photocrosslinked bands (HP) are indicated. Figure 6. Antigen immunoprecipitation of photocrosslinked antibody in human sera. Photoactivatable C3PG4m-beads were prepared as described in the scheme and incubated with HER2-hmAbs in the presence (+) or absence (-) of UV irradiation. The hmAb beads were further incubated with the HER2 proteins either in human sera (+) or 2% BSA-PBS (-). The bead bound proteins eluted by treatment of 1% SDS were subjected to reducing SDSPAGE. The protein bands on the gel were either stained with coomassie brilliant blue (CBB) or transferred to a nitrocellulose membrane for western blot (WB) analysis of the HER2 bands. The percent HER2 immunoprecipitation yields are indicated. Figure 7. Detection of EGFR proteins in human sera using EGFR-hmAb arrays irradiated UV. Antibody arrays were fabricated as described in the scheme. The amounts of hmAbs on the spots were adjusted by controlling the spotted amounts of 2xC3PG4m proteins as described in the Experimental Section. Fluorescence images depending on the EGFR concentrations were monitored by immunostaining using fluorescein-labeled anti-EGFR rat antibody (fαEGFR) at 488 nm excitation and 501-560 nm emission. Fluorescence intensities (A.U.) of the spots were calculated from the fluorescence images.



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“For TOC only”


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Covalent and Oriented Surface Immobilization of Antibody Using

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