Affinity tag systems, possessing high affinity and specificity, are useful for protein detection and purification. The most suitable tag for a particular purpose should be selected from many available affinity tag systems. In this study, we developed a novel affinity tag called the “RAP tag” system, which comprises a mouse antirat podoplanin monoclonal antibody (clone PMab-2) and the RAP tag (DMVNPGLEDRIE). This system is useful not only for protein detection in Western blotting, flow cytometry, and sandwich enzyme-linked immunosorbent assay, but also for protein purification.
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References
1.
SmithDB, and JohnsonKS: Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene, 1988; 67:31–40.
2.
DiguanC, LiP, RiggsPD, and InouyeH: Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene, 1988; 67:21–30.
3.
KatoY, KanekoMK, KunitaA, ItoH, KameyamaA, OgasawaraS, MatsuuraN, HasegawaY, Suzuki-InoueK, InoueO, OzakiY, and NarimatsuH: Molecular analysis of the pathophysiological binding of the platelet aggregation-inducing factor podoplanin to the C-type lectin-like receptor CLEC-2. Cancer Sci, 2008; 99:54–61.
4.
WangS, and HazelriggT: Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature, 1994; 369:400–403.
5.
HoppTP, PrickettKS, PriceVL, LibbyRT, MarchCJ, CerrettiDP, UrdalDL, and ConlonPJ: A short polypeptide marker sequence useful for recombinant protein identification and purification. Nat Biotechnol, 1988; 6:1204–1210.
6.
TabataS, NampoM, MiharaE, Tamura-KawakamiK, FujiiI, and TakagiJ: A rapid screening method for cell lines producing singly-tagged recombinant proteins using the “TARGET tag” system. J Proteomics, 2010; 73:1777–1785.
7.
FujiiY, KanekoM, NeyazakiM, NogiT, KatoY, and TakagiJ: PA tag: A versatile protein tagging system using a super high affinity antibody against a dodecapeptide derived from human podoplanin. Protein Expr Purif, 2014; 95:240–247.
8.
FujiiY, MatsunagaY, ArimoriT, KitagoY, OgasawaraS, KanekoMK, KatoY, and TakagiJ: Tailored placement of a turn-forming PA tag into the structured domain of a protein to probe its conformational state. J Cell Sci, 2016; 129:1512–1522.
FujiiY, OgasawaraS, OkiH, LiuX, KanekoMK, TakanoS, and KatoY: A high-sensitive HMab-2 specifically detects IDH1-R132H, the most common IDH mutation in gliomas. Biochem Biophys Res Commun, 2015; 466:733–739.
11.
LiuX, OgasawaraS, KanekoMK, OkiH, HozumiY, GotoK, TakagiM, and KatoY: A novel monoclonal antibody SMab-2 recognizes endogenous IDH2-R172S of chondrosarcoma. Biochem Biophys Res Commun, 2015; 459:636–642.
12.
Kato KanekoM, LiuX, OkiH, OgasawaraS, NakamuraT, SaidohN, TsujimotoY, MatsuyamaY, UrunoA, SugawaraM, TsuchiyaT, YamakawaM, YamamotoM, TakagiM, and KatoY: Isocitrate dehydrogenase mutation is frequently observed in giant cell tumor of bone. Cancer Sci, 2014; 105:744–748.
13.
LiuX, KatoY, KanekoMK, SugawaraM, OgasawaraS, TsujimotoY, NaganumaY, YamakawaM, TsuchiyaT, and TakagiM: Isocitrate dehydrogenase 2 mutation is a frequent event in osteosarcoma detected by a multi-specific monoclonal antibody MsMab-1. Cancer Med, 2013; 2:803–814.
KatoY, and KanekoMK: Generation of a novel monoclonal antibody WMab-1 specific for IDH2-R172W mutation. Biochem Biophys Res Commun, 2013; 433:374–378.
16.
KanekoMK, MoritaS, TsujimotoY, YanagiyaR, NasuK, SasakiH, HozumiY, GotoK, NatsumeA, WatanabeM, KumabeT, TakanoS, and KatoY: Establishment of novel monoclonal antibodies KMab-1 and MMab-1 specific for IDH2 mutations. Biochem Biophys Res Commun, 2013; 432:40–45.
17.
FujiiY, KanekoMK, and KatoY: MAP tag: A novel tagging system for protein purification and detection. Monoclon Antib Immunodiagn Immunother, 2016; 35:293–299.
18.
OgasawaraS, KanekoMK, and KatoY: LpMab-19 recognizes sialylated O-glycan on Thr76 of human podoplanin. Monoclon Antib Immunodiagn Immunother, 2016; 35:245–253.
19.
HonmaR, KanekoMK, OgasawaraS, FujiiY, KonnaiS, TakagiM, and KatoY: Specific detection of dog podoplanin expressed in renal glomerulus by a novel monoclonal antibody PMab-38 in immunohistochemistry. Monoclon Antib Immunodiagn Immunother, 2016; 35:212–216.
20.
HonmaR, OgasawaraS, KanekoM, FujiiY, OkiH, NakamuraT, TakagiM, KonnaiS, and KatoY: PMab-44 detects bovine podoplanin in immunohistochemistry. Monoclon Antib Immunodiagn Immunother, 2016 [Epub ahead of print]; DOI: 10.1089/mab.2016.0016.
21.
HochuliE, BannwarthW, DobeliH, GentzR, and StuberD: Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Nat Biotechnol, 1988; 6:1321–1325.
22.
FieldJ, NikawaJ, BroekD, MacdonaldB, RodgersL, WilsonIA, LernerRA, and WiglerM: Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol, 1988; 8:2159–2165.
23.
EvanGI, LewisGK, RamsayG, and BishopJM: Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol, 1985; 5:3610–3616.
24.
OkiH, HonmaR, OgasawaraS, FujiiY, LiuX, TakagiM, KanekoMK, and KatoY: Development of sensitive monoclonal antibody PMab-2 against rat podoplanin. Monoclon Antib Immunodiagn Immunother, 2015; 34:396–403.
25.
KatoY, FujitaN, KunitaA, SatoS, KanekoM, OsawaM, and TsuruoT: Molecular identification of Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors. J Biol Chem, 2003; 278:51599–51605.
26.
KatoY, SasagawaI, KanekoM, OsawaM, FujitaN, and TsuruoT: Aggrus: A diagnostic marker that distinguishes seminoma from embryonal carcinoma in testicular germ cell tumors. Oncogene, 2004; 23:8552–8556.
27.
KatoY, KanekoM, SataM, FujitaN, TsuruoT, and OsawaM: Enhanced expression of Aggrus (T1alpha/podoplanin), a platelet-aggregation-inducing factor in lung squamous cell carcinoma. Tumor Biol, 2005; 26:195–200.
28.
KatoY, KanekoMK, KunoA, UchiyamaN, AmanoK, ChibaY, HasegawaY, HirabayashiJ, NarimatsuH, MishimaK, and OsawaM: Inhibition of tumor cell-induced platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating domain. Biochem Biophys Res Commun, 2006; 349:1301–1307.
29.
KatoY, VaidyanathanG, KanekoMK, MishimaK, SrivastavaN, ChandramohanV, PegramC, KeirST, KuanCT, BignerDD, and ZalutskyMR: Evaluation of anti-podoplanin rat monoclonal antibody NZ-1 for targeting malignant gliomas. Nucl Med Biol, 2010; 37:785–794.
30.
KatoY, and KanekoMK: A cancer-specific monoclonal antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep, 2014; 4:5924.
31.
KatoY, KunitaA, AbeS, OgasawaraS, FujiiY, OkiH, FukayamaM, NishiokaY, and KanekoMK: The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity. Oncotarget, 2015; 6:36003–36018.
32.
KatoY, OgasawaraS, OkiH, GoichbergP, HonmaR, FujiiY, and KanekoMK: LpMab-12 established by CasMab technology specifically detects sialylated O-glycan on Thr52 of platelet aggregation-stimulating domain of human podoplanin. PLoS One, 2016; 11:e0152912.
33.
KatoY, OgasawaraS, OkiH, HonmaR, TakagiM, FujiiY, NakamuraT, SaidohN, KannoH, UmetsuM, KamataS, KuboH, YamadaM, SawaY, MoritaK, HaradaH, SuzukiH, and KanekoMK: Novel monoclonal antibody LpMab-17 developed by CasMab technology distinguishes human podoplanin from monkey podoplanin. Monoclon Antib Immunodiagn Immunother, 2016; 35:109–116.
34.
SatoC, KimJH, AbeY, SaitoK, YokoyamaS, and KohdaD: Characterization of the N-oligosaccharides attached to the atypical Asn-X-Cys sequence of recombinant human epidermal growth factor receptor. J Biochem, 2000; 127:65–72.
35.
WatanabeM, TerasawaK, KaneshiroK, UchimuraH, YamamotoR, FukuyamaY, ShimizuK, SatoTA, and TanakaK: Improvement of mass spectrometry analysis of glycoproteins by MALDI-MS using 3-aminoquinoline/alpha-cyano-4-hydroxycinnamic acid. Anal Bioanal Chem, 2013; 405:4289–4293.
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