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標題: 果糖氨基酸氧化酶表現於不同建構與大腸桿菌宿主之研究
Study on the expression of fructosyl amino acid oxidase via different constructs and Escherichia coli hosts
作者: 張晏誠
Yan-Cheng Chang
關鍵字: 果糖氨基酸氧化酶
fructosyl amino acid oxidase
SOS response
inclusion body
引用: 1. Ferri, S., et al., Review of Fructosyl Amino Acid Oxidase Engineering Research: A Glimpse into the Future of Hemoglobin A1c Biosensing. Journal of Diabetes Science and Technology, 2009. 3(3): p. 585-592. 2. Hirokawa, K., K. Nakamura, and N. Kajiyama, Enzymes used for the determination of HbA1C. FEMS Microbiol Lett, 2004. 235(1): p. 157-62. 3. Janion, C., Inducible SOS Response System of DNA Repair and Mutagenesis in Escherichia coli. International Journal of Biological Sciences, 2008. 4(6): p. 338-344. 4. Guariguata, L., et al., Global estimates of diabetes prevalence for 2013 and projections for 2035. Diabetes Research and Clinical Practice, 2014. 103(2): p. 137-149. 5. Classification and Diagnosis of Diabetes: Standards of Medical Care in Diabetes—2018. Diabetes Care, 2018. 41(Supplement 1): p. S13-S27. 6. 羅氏可霸斯糖化血紅素A1C第三代生化檢驗試劑. 2013. 7. Rosano, G.L. and E.A. Ceccarelli, Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology, 2014. 5: p. 1-17. 8. Sakaue, R., et al., Cloning and expression of fructosyl-amino acid oxidase gene from Corynebacterium sp. 2-4-1 in Escherichia coli. Bioscience, Biotechnology, and Biochemistry, 2002. 66(6): p. 1256-1261. 9. NOBUYUKI YOSHIDA, Y.S., MASAKI SERATA, YOSHIKI TANI, AND NOBUO KATO, Distribution and properties of fructosyl amino acid oxidase in fungi. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1995. 61(12): p. 4487-4489. 10. Bunn, H.F., K.H. Gabbay, and P.M. Gallop, The glycosylation of hemoglobin-relevance to diabetes mellitus. Science, 1978. 200(7): p. 21-27. 11. Honda, K., Industrial Applications of Multistep Enzyme Reactions, in Biotechnology of Microbial Enzymes. 2017. p. 433-450. 12. Keil, P., H.B. Mortensen, and C. Christophersen, Fructosylvaline. A Simple Model of the N-Terminal Residue of Human Haemoglobin A1c. Acta Chemica Scandinavica B 1985. 39: p. 191-193. 13. Wang, J., et al., Electrospray positive ionization tandem mass spectrometry of Amadori compounds. J Mass Spectrom, 2008. 43(2): p. 262-4. 14. Takahashi, N. and K. Kita, Fructosyl-Valine Orally Administrated to Chickens is Absorbed from Gastrointestinal Tract. Japan Poultry Science Association, 2016. 53: p. 153-156. 15. Pundir, C.S. and S. Chawla, Determination of glycated hemoglobin with special emphasis on biosensing methods. Analytical Biochemistry, 2014. 444: p. 47-56. 16. Horiuchi, T., T. Kurokawa, and N. Saito, Purification and Properties of Fructosyl-amino Acid Oxidase from Corynebacterium sp. 2-4-1. Agricultural and Biological Chemistry, 1989. 53(1): p. 103-110. 17. Hirokawa, K. and N. Kajiyama, Recombinant Agrobacterium AgaE-like Protein with Fructosyl Amino Acid Oxidase Activity. Biosci. Biotechnol. Biochem., 2002. 66(11): p. 2323-2329. 18. Horiuchi, T. and T. Kurokawa, Purification and Properties of Fructosylamine Oxidase from Aspergillus sp. 1005. Agricultural and Biological Chemistry, 1991. 55(2): p. 333-338. 19. Sakai, Y., et al., Purification and properties of fructosyl lysine oxidase from Fusarium oxysporum S-1F4. Biosci Biotechnol Biochem, 1995. 59(3): p. 487-491. 20. Sode, K., F. Ishimura, and W. Tsugawa, Screening and characterization of fructosyl-valine-utilizing marine microorganisms. Marine Biotechnology, 2001. 3(2): p. 126-132. 21. Martinez-Romero, E. Rhizobium tropici CIAT 899 plasmid pRtrCIAT899c, complete sequence - Nucleotide - NCBI. 2012 [cited 2018 May 28]; Available from: 22. Little, J.W. and D.W. Mount, The SOS regulatory system of Escherichia coli. Cell, 1982. 29: p. 11-22. 23. Chen, J., et al., Whole-Genome Sequence of Phage-Resistant Strain Escherichia coli DH5α. Genome Announcements, 2018. 6(10). 24. Goffin, P. and P. Dehottay, Complete Genome Sequence of Escherichia coli BLR(DE3), a recA-Deficient Derivative of E. coli BL21(DE3). Genome Announcements, 2017. 5(22). 25. Jeong, H., H.J. Kim, and S.J. Lee, Complete Genome Sequence of Escherichia coli Strain BL21. Genome Announcements, 2015. 3(2). 26. Grenier, F., et al., Complete Genome Sequence of Escherichia coli BW25113. Genome Announcements, 2014. 2(5). 27. Riley, M., et al., Escherichia coli K-12: a cooperatively developed annotation snapshot—2005. Nucleic Acids Research, 2006. 34(1): p. 1-9. 28. Wilkinson, A., et al., Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA). Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2005. 1749(1): p. 113-122. 29. Wingfield, P., Protein precipitation using ammonium sulfate. Curr Protoc Protein Sci, 2001. Appendix 3: p. Appendix 3F.
摘要: 糖化血紅素 (HbA1c) 是獲得糖尿病治療效果的重要指標[1]。果糖氨基酸氧化酶 (Fructosyl amino acid oxidase, FAOD) 已被證實是有效檢測HbA1c濃度的的酵素,有助於監測長期血糖濃度[2]。以大腸桿菌 (E. coli) 作為宿主是一種簡便方法來生產重組FAOD。本研究使用了三種宿主,BL21 (DE3)、BW25113和W3110,並且使用了兩種啟動子、商業化的T7系統和SOS調控系統[3]。我們建構了帶有T7系統的pET26b-FAOD / BL21 (DE3),SOS系統的pSOS-FAOD / BW25113與pSOS-FAOD / W3110。 在SDS-PAGE分析可以發現T7系統生產的FAOD蛋白量比SOS系統來得多,但是大部分都是沒有活性的包涵體 (Inclusion body, IB)。FAOD的分子量約為40 kDa,與宿主可溶的原生蛋白重疊。因此,使用西方墨點法 (Western blot) 分析FAOD表現量,並且利用Image J比較FAOD的表現在可溶蛋白 (supernatant) 與不可溶蛋白 (pellet) 的比例。我們發現pET26b-FAOD / BL21 (DE3)在15℃的低誘導溫度與0.01 mM IPTG的誘導下可溶FAOD可達32%。pSOS-FAOD / BW25113與pSOS-FAOD / W3110在15℃的低誘導溫度與較高濃度的0.4 μg/ml mitomycin C的誘導下可溶FAOD可達60~80%。然而,目標蛋白的表現量不高。
Glycated hemoglobin A1c (HbA1c) is an important indicator to access the effectiveness of diabetes treatment[1]. Fructosyl amino acid oxidase (FAOD), an effective enzyme helps to monitor the long term blood sugar level via the detection the concentration of HbA1c[2]. The use of recombinant Escherichia coli is a convenient way for recombinant FAOD production. Three kinds of hosts, E. coli BL21 (DE3), BW25113, and W3110 were used. Two kinds of promoters, commercial T7 system, and SOS response system were adopted. The constructs of T7 system pET26b-FAOD / BL21 (DE3), SOS response system pSOS-FAOD / BW25113, and pSOS-FAOD / W3110 were prepared. In the SDS-PAGE analysis, the T7 system produced more FAOD than SOS system, but it contained lots of inclusion body with no activity. The molecular weight of FAOD is approximately 40 kDa, where it overlaps with the host's soluble protein. Therefore, western blot was used for FAOD analysis. Image J was applied to evaluate the protein level between supernatant and pellet. We found pET26b-FAOD / BL21 (DE3) expressed 32% soluble FAOD under low induction temperature of 15℃and 0.01 mM IPTG. The pSOS-FAOD / BW25113, and pSOS-FAOD / W3110 expressed 60-80% soluble FAOD under low induction temperature of 15℃and 0.4 μg/ml mitomycin C. However, the target protein was expressed in a low level.
文章公開時間: 2021-08-17
Appears in Collections:化學工程學系所



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