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11 (1) 2021

Cloning, expression and purification of fructosyl amino acid oxidase (FAOX) in Escherichia Coli


Author - Affiliation:
Ho Ta Giap - Nguyen Tat Thanh University, Vietnam
Phan Ngoc Han - Nguyen Tat Thanh University, Vietnam
Tran Le Duy Phuong - Nguyen Tat Thanh University, Vietnam
Phung Thi Thu Phung - Nguyen Tat Thanh University, Vietnam
Vu Van Van - Nguyen Tat Thanh University, Vietnam
Corresponding author: htgiap@ntt.edu.vn
DOI:10.46223/HCMCOUJS.tech.en.11.1.1238.2021

Abstract
Introduction: The level of serum HbA1c is an indicator of the average blood sugar level in the last three months. HbA1c can be quantified using assays involving the enzyme fructosyl amino acid oxidase (FAOX). This study aims to produce GST-tagged FAOX-TE (GST/FAOX-TE), a thermal stable and specific variant of FAOX, for future application studies.
Materials and methods: The E. coli strains DH5α and BL21 (DE3) were used as cloning and expression hosts, respectively. The FAOX-TE sequence was synthesized at IDT (US) and clonned into pGEX-4T3 vector, which was confirmed by Colony PCR. The expression was induced at 16°C, 0.5 mM IPTG in LB media containing 50 µg/ml ampicilin. The protein expression profile was analyzed by SDS-PAGE. The cell pellet was sonicated and purified by Glutathione Sepharose 4 Fast Flow (Cytiva, US). The catalytic activity of GST/FAOX-TE with fructosyl valine was determined using high performance anion exchange chromatography with pulsed amperometry detection (HPAEC-PAD).
Results: The fusion protein was successfully expressed in Escherichia coli using the plasmid pGEX-4T3 and purified to high purity 93%. Recombinant GST/FAOX-TE was shown to be active on fructosyl valine.
Conclusions: Active GST/FAOX-TE was successfully expressed in E. coli BL21 (DE3) and purified, which will be used for future development of biosensors for fructosyl valine quantification.

Keywords
fructosylamino acid oxidase; FAOX-TE; expression and purification

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References

Akazawa, S., Karino, T., Yoshida, N., Katsuragi, T., & Tani, Y. (2004). Functional analysis of fructosyl-amino acid oxidases of aspergillus oryzae. Applied and Environmental Microbiology, 70(10), 5882-5890. doi:10.1128/AEM.70.10.5882-5890.2004


Bunn, H. F., Haney, D. N., Kamin, S., Gabbay, K. H., & Gallop, P. M. (1976). The biosynthesis of human hemoglobin A1c. Slow glycosylation of hemoglobin in vivo. Journal of Clinical Investigation, 57(6), 1652-1659.


Ferri, S., Kim, S., Tsugawa, W., & Sode, K. (2009). Review of fructosyl amino acid oxidase engineering research: A glimpse into the future of hemoglobin A1c biosensing. Journal of Diabetes Science and Technology, 3(3), 585-592. doi:10.1177/193229680900300324


Ferri, S., Sakaguchi, A., Goto, H., Tsugawa, W., & Sode, K. (2005). Isolation and characterization of a fructosyl-amine oxidase from an arthrobacter sp. Biotechnology Letters, 27(1), 27-32. doi:10.1007/s10529-004-6312-z


Hirokawa, K., Gomi, K., Bakke, M., & Kajiyama, N. (2003). Distribution and properties of novel deglycating enzymes for fructosyl peptide in fungi. Archives of Microbiology, 180(3), 227-231. doi:10.1007/s00203-003-0584-x


Hirokawa, K., & Kajiyama, N. (2002). Recombinant agrobacterium AgaE-like protein with fructosyl amino acid oxidase activity. Bioscience, Biotechnology, and Biochemistry, 66(11), 2323-2329. doi:10.1271/bbb.66.2323


Keil, P., Mortensen, H. B., & Christophersen, C. (1985). Fructosylvaline: A simple model of the N-terminal residue of human haemoglobin A1c. Acta Chemica Scandinavica. Series B: Organic Chemistry and Biochemistry, 39(3), 191-193. doi:10.3891/acta.chem.scand.39b-0191


Rajkumar, R., Warsinke, A., Möhwald, H., Scheller, F. W., & Katterle, M. (2007). Development of fructosyl valine binding polymers by covalent imprinting. Biosensors and Bioelectronics, 22(12), 3318-3325. doi:10.1016/j.bios.2007.03.001


Sakai, Y., Yoshida, N., Isogai, A., Tani, Y., & Kato, N. (1995). Purification and properties of fructosyl lysine oxidase from Fusarium oxysporum S-1F4. Bioscience, Biotechnology, and Biochemistry, 59(3), 487-491. doi:10.1271/bbb.59.487


Sakai, Y., Yoshida, N., Tani, Y., & Kato, N. (1996). Production of fructosyl lysine oxidase from Fusarium oxysporum S-1F4 on autoclave-browned medium. Bioscience, Biotechnology, and Biochemistry, 60(1), 150-151. doi:10.1271/bbb.60.150


Sakaue, R., & Kajiyama, N. (2003). Thermostabilization of bacterial fructosyl-amino acid oxidase by Directed evolution. Applied and Environmental Microbiology, 69(1), 139-145. doi:10.1128/AEM.69.1.139-145.2002


Sakurabayashi, I., Watano, T., Yonehara, S., Ishimaru, K., Hirai, K., Komori, T., & Yagi, M. (2003). New enzymatic assay for glycohemoglobin. Clinical Chemistry, 49(2), 269-274. doi:10.1373/49.2.269


Sode, K., Ishimura, F., & Tsugawa, W. (2001). Screening and characterization of fructosyl-valine-utilizing marine microorganisms. Marine Biotechnology (New York, N.Y.), 3(2), 126-132. doi:10.1007/s101260000065


Takahashi, M., Pischetsrieder, M., & Monnier, V. M. (1997a). Isolation, purification, and characterization of amadoriase isoenzymes (fructosyl amine-oxygen oxidoreductase EC 1.5.3) from Aspergillus sp. The Journal of Biological Chemistry, 272(6), 3437-3443. doi:10.1074/jbc.272.6.3437


Takahashi, M., Pischetsrieder, M., & Monnier, V. M. (1997b). Molecular cloning and expression of amadoriase isoenzyme (fructosyl amine:oxygen oxidoreductase, EC 1.5.3) from Aspergillus fumigatus. The Journal of Biological Chemistry, 272(19), 12505-12507. doi:10.1074/jbc.272.19.12505


Wu, X., Takahashi, M., Chen, S. G., & Monnier, V. M. (2000). Cloning of amadoriase I isoenzyme from Aspergillus sp: Evidence of FAD covalently linked to Cys342. Biochemistry, 39(6), 1515-1521. doi:10.1021/bi992031g


Yari, K., Afzali, S., Mozafari, H., Mansouri, K., & Mostafaie, A. (2013). Molecular cloning, expression and purification of recombinant soluble mouse endostatin as an anti-angiogenic protein in Escherichia coli. Molecular Biology Reports, 40(2), 1027-1033. doi:10.1007/s11033-012-2144-4


Yoshida, N., Sakai, Y., Isogai, A., Fukuya, H., Yagi, M., Tani, Y., & Kato, N. (1996). Primary structures of fungal fructosyl amino acid oxidases and their application to the measurement of glycated proteins. European Journal of Biochemistry, 242(3), 499-505. doi:10.1111/j.1432-1033.1996.0499r.x


Yoshida, N., Sakai, Y., Serata, M., Tani, Y., & Kato, N. (1995). Distribution and properties of fructosyl amino acid oxidase in fungi. Applied and Environmental Microbiology, 61(12), 4487-4489.



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