類澱粉蛋白(Amyloid protein)具高度自組裝性，可自組裝形成類澱粉蛋白纖維(Amyloid fiber)，在大腸桿菌(E.coli)中之類澱粉蛋白CsgA則可自組裝形成捲曲纖維(Curli fiber)，用以幫助大腸桿菌附著於各種不同材料表面，此捲曲纖維具有高度化學穩定性且能抗蛋白酶水解，被廣泛應用於各種酵素之固定化，果糖胜肽氧化酶(Fructosyl peptide oxidase, FPOX)是一種用於呈色法快速檢測糖化血紅素HbA1c之酵素，糖化血紅素則是可作為近2~3個月平均血糖之指標，一般常做為糖尿病患控制血糖之依據。SpyTag/ SpyCatcher是一種被廣泛使用的特異性功能標籤胜肽與蛋白，其兩者結合後能形成不可逆之醯胺鍵，將兩酵素或蛋白結合形成多功能性蛋白。
本論文研究以基因工程方法，建構兩個融合蛋白分別為CsgA與SpyTag融合之蛋白，利用CsgA易於在表面自組裝形成類澱粉蛋白纖維來做為固定化酵素之載體蛋白，及FPOX與SpyCatcher融合蛋白，利用已附著在固體表面之CsgA融合SpyTag，來接附含有FPOX之SpyCatcher，而將FPOX固定化，可應用於檢測血液中之糖化血紅素含量。在大腸桿菌中表現純化後之SpyCatcher-FPOX融合蛋白其活性與另一融合蛋白CBD-FPOX酵素相比，其FPOX活性下降約31%，在與CsgA-SpyTag蛋白接合反應後，仍然保持FPOX活性，但其活性隨溫度上升而下降，在35℃與40℃時因接合固定化而穩定其活性，而使活性分別可增加19%與9%。

Amyloid protein is a highly self-assembling protein, easy to assemble into insoluble fiber. The amyloid-like CsgA protein secrets from E. coli can self-assemble to form curli fiber, which can help E. coli cells attach onto the surface of various materials. The curli fiber is highly stable and resistant to protease hydrolysis. Fructosyl peptide oxidase (FPOX) is the key enzyme used in the colorimetric method for detecting glycated hemoglobin HbA1c. HbA1c often used as an indicator of average blood glucose in the past 2 to 3 months. Its value is used for blood glucose control in diabetic patients. SpyTag/ SpyCatcher is recently discovered specific covalent bioconjugation system. The separately produced tag peptide and catcher protein, when in contact with each other will spontaneously form an irreversible amide bonding.
In this work, CsgA protein was first expressed and its formation as curli fiber was characterized. CsgA was then fused with SpyTag and FPOX was fused with SpyCatcher. By taking advantage of CsgA’s amyloid-like fibers formation, the fused SpyTag was used to immobilize SpyCatcher fused FPOX on the amyloid fibers. Compare with CBD fused FPOX (CBD-FPOX), FPOX activity of SpyCatcher-FPOX fusion protein reduced about 31%. After conjugation with CsgA-SpyTag protein, FPOX activity still could be maintained. At 35℃ and 40℃, its activity increased by 19% and 9%, respectively due to the effect of bioconjugation immobilization.
 

Axpe, E., Duraj-Thatte, A., Chang, Y., Kaimaki, D.-M., Sanchez-Sanchez, A., Caliskan, H. B., . . . Joshi, N. S. (2018). Fabrication of amyloid curli fibers–alginate nanocomposite hydrogels with enhanced stiffness. ACS Biomaterials Science & Engineering, 4(6), 2100-2105.
Barnhart, M. M., & Chapman, M. R. (2006). Curli biogenesis and function. Annu Rev Microbiol, 60, 131-147. doi: 10.1146/annurev.micro.60.080805.142106
Biancalana, M., & Koide, S. (2010). Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1804(7), 1405-1412. doi: https://doi.org/10.1016/j.bbapap.2010.04.001
Botyanszki, Z., Tay, P. K., Nguyen, P. Q., Nussbaumer, M. G., & Joshi, N. S. (2015). Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers. Biotechnol Bioeng, 112(10), 2016-2024. doi: 10.1002/bit.25638
Chapman, M. R., Robinson, L. S., Pinkner, J. S., Roth, R., Heuser, J., Hammar, M., . . . Hultgren, S. J. (2002). Role of Escherichia coli curli operons in directing amyloid fiber formation. Science, 295(5556), 851-855.
DeBenedictis, E. P., Liu, J., & Keten, S. (2016). Adhesion mechanisms of curli subunit CsgA to abiotic surfaces. Science advances, 2(11), e1600998.
Deivanayagam, C. C., Rich, R. L., Carson, M., Owens, R. T., Danthuluri, S., Bice, T., . . . Narayana, S. V. (2000). Novel fold and assembly of the repetitive B region of the Staphylococcus aureus collagen-binding surface protein. Structure, 8(1), 67-78.
Dong, H., Zhang, W., Xuan, Q., Zhou, Y., Zhou, S., Huang, J., & Wang, P. (2021). Binding Peptide-Guided Immobilization of Lipases with Significantly Improved Catalytic Performance Using Escherichia coli BL21(DE3) Biofilms as a Platform. ACS Appl Mater Interfaces, 13(5), 6168-6179. doi: 10.1021/acsami.0c18298
Dorval Courchesne, N. M., Duraj-Thatte, A., Tay, P. K. R., Nguyen, P. Q., & Joshi, N. S. (2017). Scalable Production of Genetically Engineered Nanofibrous Macroscopic Materials via Filtration. ACS Biomater Sci Eng, 3(5), 733-741. doi: 10.1021/acsbiomaterials.6b00437
Evans, M. L., Chorell, E., Taylor, J. D., Åden, J., Götheson, A., Li, F., . . . Wittung-Stafshede, P. (2015). The bacterial curli system possesses a potent and selective inhibitor of amyloid formation. Molecular cell, 57(3), 445-455.
Goldberg, M. S., & Lansbury Jr, P. T. (2000). Is there a cause-and-effect relationship between α-synuclein fibrillization and Parkinson’s disease? Nature Cell Biology, 2(7), E115-E119. doi: 10.1038/35017124
Hammar, M. r., Arnqvist, A., Bian, Z., Olsén, A., & Normark, S. (1995). Expression of two csg operons is required for production of fibronectin‐and congo red‐binding curli polymers in Escherichia coli K‐12. Molecular microbiology, 18(4), 661-670.
Hirokawa, K., Gomi, K., & Kajiyama, N. (2003). Molecular cloning and expression of novel fructosyl peptide oxidases and their application for the measurement of glycated protein. Biochem Biophys Res Commun, 311(1), 104-111. doi: 10.1016/j.bbrc.2003.09.169
Hirokawa, K., Shimoji, K., & Kajiyama, N. (2005). An enzymatic method for the determination of hemoglobinA 1C. Biotechnology letters, 27(14), 963-968.
Hoffner, G., & Djian, P. (2015). Polyglutamine aggregation in Huntington disease: does structure determine toxicity? Molecular neurobiology, 52(3), 1297-1314.
Howie, A. J., & Brewer, D. B. (2009). Optical properties of amyloid stained by Congo red: history and mechanisms. Micron, 40(3), 285-301.
Jain, N., Ådén, J., Nagamatsu, K., Evans, M. L., Li, X., McMichael, B., . . . Chapman, M. R. (2017). Inhibition of curli assembly and Escherichia coli biofilm formation by the human systemic amyloid precursor transthyretin. Proceedings of the National Academy of Sciences, 114(46), 12184-12189.
Kaushik, A., Sharma, S. K., Chatzinotas, S., Ottersten, B., & Jondral, F. K. (2016). On the performance analysis of underlay cognitive radio systems: A deployment perspective. IEEE Transactions on Cognitive Communications and Networking, 2(3), 273-287.
Kim, S., Ferri, S., Tsugawa, W., Mori, K., & Sode, K. (2010). Motif-based search for a novel fructosyl peptide oxidase from genome databases. Biotechnol Bioeng, 106(3), 358-366. doi: 10.1002/bit.22710
Lin, Y., Jin, W., Wang, J., Cai, Z., Wu, S., & Zhang, G. (2018). A novel method for simultaneous purification and immobilization of a xylanase-lichenase chimera via SpyTag/SpyCatcher spontaneous reaction. Enzyme Microb Technol, 115, 29-36. doi: 10.1016/j.enzmictec.2018.04.007
Nanjo, Y., Hayashi, R., & Yao, T. (2007). An enzymatic method for the rapid measurement of the hemoglobin A1c by a flow-injection system comprised of an electrochemical detector with a specific enzyme-reactor and a spectrophotometer. Anal Chim Acta, 583(1), 45-54. doi: 10.1016/j.aca.2006.09.037
Nguyen, P. Q., Botyanszki, Z., Tay, P. K., & Joshi, N. S. (2014). Programmable biofilm-based materials from engineered curli nanofibres. Nat Commun, 5, 4945. doi: 10.1038/ncomms5945
Onur, T., Yuca, E., Olmez, T. T., & Seker, U. O. S. (2018). Self-assembly of bacterial amyloid protein nanomaterials on solid surfaces. Journal of colloid and interface science, 520, 145-154.
Reddington, S. C., & Howarth, M. (2015). Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr Opin Chem Biol, 29, 94-99. doi: 10.1016/j.cbpa.2015.10.002
Sleutel, M., Van den Broeck, I., Van Gerven, N., Feuillie, C., Jonckheere, W., Valotteau, C., . . . Remaut, H. (2017). Nucleation and growth of a bacterial functional amyloid at single-fiber resolution. Nat Chem Biol, 13(8), 902-908. doi: 10.1038/nchembio.2413
Taglialegna, A., Lasa, I., & Valle, J. (2016). Amyloid Structures as Biofilm Matrix Scaffolds. J Bacteriol, 198(19), 2579-2588. doi: 10.1128/JB.00122-16
Zakeri, B., Fierer, J. O., Celik, E., Chittock, E. C., Schwarz-Linek, U., Moy, V. T., & Howarth, M. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A, 109(12), E690-697. doi: 10.1073/pnas.1115485109
巫宜庭. (2016). 纖維素結合功能域(CBD)融合醣化胜肽氧化酶(FPOX)之表現與其在測定糖化血紅素(HbA1c)之應用. (碩士), 國立臺灣科技大學, 台北市. Retrieved from https://hdl.handle.net/11296/g5xv99