Evidence for Multiple Applications of Monoclonal Antibody 5G10

Cell and plasmids

Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT) and penicillin/streptomycin at 37°C in a 5% carbon dioxide humidified atmosphere. Plasmid-containing Japanese encephalitis virus (JEV; SA14-14-2 strain) NS5-MTase gene was a gift from Dr. Cao of Huazhong Agricultural University (HZAU), China.⁽⁸⁾ pEF-BO-FLAG-MAVS was provided by Dr. Luo of HZAU, China.⁽⁹⁾

Fluorescent-activated cell sorting analysis

After 48 h transfection with plasmids pIRES-T5-2A, pIRES-2A-T5 or pIRES, 1 × 10 ⁵ 293T cells were stained with 5G10 antibody or isotype control 4D1⁽¹⁰⁾ at 4°C for 30 minutes. After washing three times with phosphate-buffered saline containing 1% FBS, the 293T cells were restained with fluorescein (FITC)-labeled goat antimouse immunoglobulin G (IgG) antibody. After washing three times, the 293T cells were analyzed by fluorescent-activated cell sorting (FACS).

Purification of 2A and NS5 methyltransferase

Purified 5G10 antibody (5 mg) was immobilized in a 1 mL HiTrap column (Amersham Pharmacia Biotech) according to the instructions of the manufacturer. The gel in the HiTrap column consisted of highly cross-linked agarose beads, activated by N-hydroxysuccinimide to allow coupling of ligands that contain primary amino groups. Ten 10 cm plates of 293T cells were transfected with pIRES-2A-T5 or pCAGGS-JEV NS5-T5 methyltransferase (MTase)-T5. Forty-eight hours after transfection, cells were lysed and allowed to pass through a column harboring 5G10 agarose beads. The beads were then washed with 80 mL of Tris-buffered saline (pH 7.5) and eluted with 0.1 mg/mL of epitope peptide QRVRELAV.

Immunoprecipitation

Forty-eight hours after transfection with plasmids pIRES-T5-2A, pIRES-2A-T5, or pIRES, cells were lysed with a radioimmunoprecipitation assay (25 mM Tris-HCl buffer [pH 7.4] containing 150 mM sodium chloride, 1% NP-40, 0.25% sodium deoxycholate) containing a protease inhibitor cocktail (Roche, Indianapolis, IN). Cell lyates were incubated with 5G10 antibody or anti-FLAG antibody (Sigma, St. Louis, MO) at 4°C overnight on a rotator in the presence of protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitates were subjected to electrophoresis and Western blot analysis.

5G10 recognizes either the N- or C-terminal peptide QRVRELAV

We had earlier obtained a murine mAb 5G10 against pathogenic bacteria SF and commensal bacteria K12 flagellin (KF). Interestingly, mAb 5G10 impaired the engagement of flagellin and TLR5 and subsequently resulted in less expression of interleukin 8 and monocyte chemotactic protein 1.⁽⁶⁾ The linear epitope (residues 90–97 of SF, QRVRELAV) on flagellin is considered to be crucial for TLR5 activation (Fig. 1A).^(7,11) Therefore, we wanted to know whether 5G10 could identify peptide QRVRELAV (T5 tag) when the tag was fused with a target protein.

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FIG. 1.

5G10 recognizes a short epitope QRVRELAV on bacterial flagellin. (A) Crystal structure of the SF that contains conserved D0 and D1 region and hypervariable regions D2 and D3. Arrowhead indicates the domain (QRVRELAV) on flagellin for activating TLR5 signaling. (B) Schematic diagram of SF and its variants. The SF, SF90-506, and SF1-97 represented full-length flagellin (1–506 residues), a variant containing 90–506 residues and the other variant containing 1–97 residues of SF. (C) SF and its mutants were expressed in BL21 (DE3) and determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Lanes 1–4 represented BL21-containing pET28a(+) vector, SF, SF90-506, or SF1-97 after being induced by IPTG. Asterisks indicate that the corresponding proteins expressed SF 55 kDa, SF90-506 46 kDa, and SF1-97 9 kDa, respectively. (D) 5G10 recognized a short epitope QRVRELAV on SF. The proteins in (C) were transferred onto PVDF membrane and hybridized with the antibody 5G10. Western blot result showed that 5G10 recognized full-length of SF and SF mutants with the N-terminal or C-terminal T5 tag. IPTG, isopropyl-β-d-thiogalactoside; PVDF, polyvinylidene fluoride; SF, Salmonella flagellin; TLR5, Toll-like receptor 5.

To test whether the terminal T5 could be identified, recombinant plasmids were constructed to express residues 1–97 or residues 90–506 of flagellin (Fig. 1B). After being induced by isopropyl-β-d-thiogalactoside (IPTG), expression of the full-length wild-type SF and two truncated versions of SF (SF1-97 and SF90-506) in BL21 (DE3) was assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) assay. SF, SF1-97, and SF90-506 had putative molecular weight (MW) of ∼55, 46, and 9 kDa, respectively (Fig. 1C), which was in accordance with our expectation. Then, we detected the T5 tag in the distinct position of protein with 5G10 by Western blot assay. The result showed that the T5 tag was not only embedded in intact SF but was also exposed in either terminal of SF variants and both could be recognized by 5G10 (Fig. 1D). Therefore, the short peptide QRVRELAV in any position of protein could be recognized by 5G10, which suggested that the T5 tag and 5G10 mAb might be utilized as potential tools for fusion protein detection.

Detecting T5-fused eukaryocytic EV71 2A by 5G10

The T5 tag in either terminal of prokaryotic flagellin could be detected by 5G10. We next set enterovirus 71 (EV71 BrCr strain) nonstructural protein 2A and T5 tag as fusion eukaryocytic protein example to test the binding activity of 5G10 (Fig. 2A). We first constructed two plasmids (designated pIRES-T5-2A and pIRES-2A-T5) that contained a 5′- or 3′-terminal tag in EV71 2A gene. The recombinant plasmids pIRES-T5-2A, pIRES-2A-T5, or pIRES control were transfected to HEK 293T cells. Forty-eight hours later, the transfected cells were harvested and subjected to different analyses, such as IFA, Western blot, and FACS. First, after transfection with the plasmids, cells were fixed and permeated. 5G10 and an isotype antibody control 4D1 were used to detect fusion proteins T5-2A and 2A-T5. The IFA results showed that the fluorescent signal in the transfected cell could be detected by 5G10 but not by the isotype antibody 4D1 (Fig. 2B). Second, the transfected cells were harvested and subjected to SDS-PAGE and the proteins were transferred onto polyvinylidene fluoride membrane. 5G10, but not the isotype control antibody 4D1, detected both fusion proteins T5-2A and 2A-T5 by Western blot assay (Fig. 2C). Third, the transfected cells were subjected to FACS assay after 5G10 dyeing. FITC-labeled goat antimouse IgG was used as the second detection antibody. 5G10 led to numerous transfected cell high fluorescence signal, but not the isotype control antibody (Fig. 2D). Statistically, medium fluorescent intensity in 5G10 dyeing groups was significantly higher than that in the isotype control antibody 4D1 (Fig. 2E) (p < 0.001). Therefore, 5G10 could be used as an alternative antibody to detect expressed eukaryotic fusion protein with a T5 tag.

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FIG. 2.

Detecting T5-fused EV71 2A by 5G10. (A) Schematic diagram of the combination manner of EV71 2A and T5 tag in recombinant plasmids. The recombinant plasmids pIRES-T5-2A, pIRES-2A-T5, or pIRES were transfected to HEK 293T cells, the transfected cells were harvested and subjected to different analyses, such as IFA, Western blot, and FACS. (B) The IFA results indicated that 5G10 bound to T5 tag expressed in HEK 293T cells. The IFA results showed that the fluorescent signal in the transfected cell could be detected by 5G10, but not by 4D1 isotype antibody. (C) The Western blot results showed that 5G10, but not the isotype control antibody 4D1, could detect both fusion proteins T5-2A and 2A-T5. (D) The FACS results showed that 5G10 labeling led to numerous transfected cells high fluorescence signal, but not the isotype control antibody 4D1. (E) MFI in 5G10 treatment group was significantly higher than that in the isotype control antibody 4D1 (***p < 0.001). FACS, fluorescent-activated cell sorting; HEK, human embryonic kidney; IFA, immunofluorescence assay; MFI, medium fluorescent intensity.

Purifying fusion proteins 2A-T5 and NS5 MTase-T5 and dragging out mitochondrial antiviral signaling protein that interacts with EV71 2A protein using 5G10

We also wanted to know whether 5G10 could be used as a bait for protein affinity purification. The 5G10 was first coupled with agarose beads as per the illustration provided by the manufacturer. pIRES-2A-T5 or pCAGGS-JEV (SA14-14-2 strain) NS5-T5 MTase transfected cells were subjected to lyse and passed through a column harboring 5G10 agarose bead. After adequate incubation, the target protein T5-2A or NS5 MTase-T5 was competitively eluted with the peptide QRVRELAV. The 2A-T5 and NS5 MTase-T5 fusion proteins with MW 17 kDa (Fig. 3A) and 65 kDa (Fig. 3B) were eluted, respectively, as shown in the gel of SDS-PAGE. The arrowhead indicates the corresponding proteins 2A-T5 (Fig. 3A) and NS5 MTase-T5 (Fig. 3B). Hence, 5G10 could also be used for affinity protein purification.

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FIG. 3.

Purification of fusion proteins 2A-T5 and NS5 MTase-T5 and dragging out MAVS protein that interacts with EV71 2A protein using 5G10. 5G10 was coupled on agarose beads according to the manufacturer illustration. pIRES-2A-T5 or pCAGGS-JEV NS5 MTase-T5-transfected cells were lysed and allowed to pass through the column containing agarose beads, and the target protein 2A-T5 or NS5 MTase-T5 was competitively eluted with peptide QRVRELAV finally. The 2A-T5 and NS5 MTase-T5 fusion proteins with MW 17 kDa (A) and 65 kDa (B) were eluted, respectively, as shown in the gel of SDS-PAGE. pEF-BO-Flag-MAVS was cotransfected with pIRES-2A-T5 or pIRES vector into HEK 293T cells; 48 hours later, the cells were subjected to Co-IP assay by 5G10 as a bait antibody. (C) Co-IP assay result showed that 5G10 could drag out the complex of 2A-T5 and its conjugating protein MAVS. Co-IP, co-immunoprecipitation; MAVS, mitochondrial antiviral signaling; MW, molecule weight.

As an eight-amino acid short peptide, the protein property of T5 tag could almost not change when fused. Thus, 5G10 could be used as a bait antibody to drag out novel proteins that are interacting with a known protein. As is known, EV71 2A binds to mitochondrial antiviral signaling (MAVS) protein and cleaves it to interrupt interferon α/β production.⁽¹²⁾ Here we attempted to assess whether MAVS protein could be pulled out during 2A-T5 baiting by 5G10. pEF-BO-FLAG-MAVS was cotransfected with pIRES-2A-T5 into HEK 293T cells, 48 hours later the cells were subjected to co-immunoprecipitation (Co-IP) assay by 5G10 as a bait antibody. The result showed that 5G10 could drag out the complex of 2A-T5 and its conjugating protein MAVS (Fig. 3C), as could a positive control antibody anti-FLAG. Thus, it demonstrated that 5G10 could be used as a bait antibody for novel protein exploration.

In summary, in this study, we provide solid evidence for applications of mAb 5G10. When fused with the short T5 tag, a target protein could be detected, traced, and even purified by 5G10. Therefore, the murine mAb 5G10 combined with the corresponding T5 tag can be used as potential tools in biological research, including protein location, novel protein exploration, and protein purification.