GATS tag system is compatible with biotin labelling methods for protein analysis

www.nature.com/scientificreports OPEN GATS tag system is compatible with biotin labelling methods for protein analysis Kohdai Yamada 1, Fumiya Soga 1, Soh Tokunaga 1, Hikaru Nagaoka 2, Tatsuhiko Ozawa 3, Hiroyuki Kishi 3, Eizo Takashima 2 & Tatsuya Sawasaki 1* Polypeptide tags and biotin labelling technologies are widely used for protein analyses in biochemistry and cell biology. However, many peptide tag epitopes contain lysine residues (or amino acids) that are masked after biotinylation. Here, we propose the GATS tag system without a lysine residue and with high sensitivity and low non-specific binding using a rabbit monoclonal antibody against Plasmodium falciparum glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (PfGAMA). From 14 monoclonal clones, an Ra3 clone was selected as it recognized an epitope—TLSVGVQNTF— without a lysine residue; this antibody and epitope tag set was called the GATS tag system. Surface plasmon resonance analysis showed that the tag system had a high affinity of 8.71 × 10–9 M. GATS tag indicated a very low background with remarkably high sensitivity and specificity in immunoblotting using the lysates of mammalian cells. It also showed a high sensitivity for immunoprecipitation and immunostaining of cultured human cells. The tag system was highly sensitive in both biotin labelling methods for proteins using NHS-Sulfo-biotin and BioID (proximity-dependent biotin identification) in the human cells, as opposed to a commercially available tag system having lysine residues, which showed reduced sensitivity. These results showed that the GATS tag system is suitable for methods such as BioID involving labelling lysine residues. Biotin labelling technology has been widely used in many studies, in the life sciences and chemistry. In particular, because N-Hydroxysuccinimide (NHS)-ester can react with the amino groups of proteins, such as lysine residue or N-terminal, NHS-biotin is available in commercial kits and has been used for biotin labelling of proteins. In addition, the BioID (proximity-dependent biotin identification) method has been widely used for protein–protein interaction (PPI) analysis. BioID technology uses proximity biotinylation enzymes such as B ioID1,2, TurboID3, and AirID4. The enzyme is fused to the protein of interest (POI) and subsequently carries out biotin labelling of the proximity proteins. These features have been used to perform comprehensive PPI in cells5 and organisms6. Recently, we developed the proximity biotinylation enzyme A irID4 and analyzed the drug-dependent P PI7. The BioID enzyme produces biotinyl-5′-AMP as an i ntermediate2, which reacts with the amino group of a side chain in the lysine residue. Therefore, lysine residues in proteins are modified by biotin. Polypeptide tag technology is used in many PPI analyses, such as co-immunoprecipitation8, AlphaScreen assay9–11, and protein array12–14. Tag systems are also used in many life science experiments, such as cell biology and transgenic organisms. In most life science studies, commercially available peptide tag systems, such as FLAG15, MYC16, and HA17, have been used to detect or analyze target proteins. Surprisingly, many tag systems, except for the HA tag, have a lysine residue in the epitope amino acid. Because the biotin labelling technology described above modifies lysine residues, many tag systems are not suitable for the functional analysis of proteins using biotin labelling. Therefore, a new tag system that excludes lysine residues is required for protein analysis using biotin labelling. In this study, we developed the GATS tag as a novel tag system that uses a rabbit monoclonal antibody against Plasmodium falciparum glycosylphosphatidylinositol (GPI)- anchored micronemal antigen (PfGAMA) localized to malarial micronemes (https://plasmodb.org/plasmo/app/record/gene/PF3D7_0828800). The GATS tag system showed a high affinity of 8.71 × 10–9 M on surface plasmon resonance (SPR) and provided high sensitivity and low background on immunoblotting using the lysates of mammalian cells. It has also been used 1 Division of Cell‑Free Life Science, Proteo-Science Center, Ehime University, 3 Bunkyo‑Cho, Matsuyama, Ehime 790‑8577, Japan. 2Division of Malaria Research, Proteo-Science Center, 3 Bunkyo‑Cho, Matsuyama, Ehime 790‑8577, Japan. 3Department of Immunology, Faculty of Medicine, Academic Assembly, Advanced Antibody Drug Development Center, University of Toyama, Toyama 930‑0194, Japan. *email: sawasaki@ ehime-u.ac.jp Scientific Reports | (2023) 13:10243 | https://doi.org/10.1038/s41598-023-36858-y 1 Vol.:(0123456789) www.nature.com/scientificreports/ for immunoprecipitation and immunostaining of cultured human cells. Furthermore, the system showed high performance in the detection of proteins in the NHS-biotin and BioID methods, whereas the FLAG tag reduced the sensitivity. This GATS tag system provides a useful tool for the functional analysis of biotin labelling proteins, such as in the BioID method. Results Isolation and characterization of rabbit monoclonal antibodies against Plasmodium falciparum GAMA protein. GAMA is a protein of the malaria parasite P. falciparum (Fig. 1a); it is known to local- ize on secretory organelles called micronemes18,19. We selected the 602aa-715aa in PfGAMA protein because it showed high protein productivity, [ GAMA602-715, (GAMA-F) Fig. 1b]. The recombinant GAMA-F protein was synthesized as a C-terminal Strep-tag fusion form by a wheat germ cell-free protein synthesis system20. The malaria protein PfRipr fragment [Ripr720-934,(Ripr-F)]21 was used as a control for specificity evaluation (https:// plasmodb.org/plasmo/app/record/gene/PF3D7_0323400). Fourteen antibody gene sets consisting of heavy and light chain genes were cloned using the immunospot array assay on a chip (ISAAC) method21, and each antibody was expressed in Expi293F cells. Specificity was evaluated by immunoblotting using the antigen GAMA-F (~ 15 kDa) (Fig. 1c). Ripr-F, a part of P. falciparum Rh5 interacting protein (PfRipr)21 was used as a control for specificity evaluation. Positive antibodies (red circles in Fig. 1c) were used in the binding assay using the AlphaScreen assay. Seven out of eight rabbits (Ra) monoclonal antibody (mAb) clones specifically recognized GAMA-F (Fig. 1d). Furthermore, three (Ra3, Ra9, and Ra13) mAbs showed a clear band in immunoblotting using lysates from P. falciparum (blood stage) (Fig. 1e). Therefore, Ra3, Ra9, and Ra13 mAbs were further analyzed. Rough epitope mappings of three anti‑ GAMA‑F antibodies. To determine the approximate epitope of Ra mAbs, GAMA-F was divided into three major fragments. The three fragments were named A, B, and C, and were further fragmented to narrow down the epitope in detail (Fig. 2a). All the fragments were synthesized by the wheat cell-free system as a fusion to the C-terminus of GST-TEV-bls and were then used to determine the binding regions of the three antibodies by (...truncated)