應用最小葡聚醣結合功能區塊作為重組蛋白質純化用之親和性標籤

Application of the Minimal Dextran-Binding Domain as an Affinity Tag for Recombinant Protein Purification

10.6578/TJACFS.201902_57(1).0005

黃文正(Wen-Zheng Huang)；陳正文(Zeng-Weng Chen)；卓侑霖(You-Lin Zhuo)；林俊宏(Jiunn-Horng Lin)；宣詩玲(Shih-Ling Hsuan)；王志鵬(Jyh-Perng Wang)

腸膜明串珠菌 ； 葡聚醣蔗糖酶 ； 葡聚醣結合功能區塊 ； 親和性標籤 ； Leuconostoc mesenteroides ； dextransucrase ； dextran-binding domain ； affinity tag

臺灣農業化學與食品科學

57卷1期（2019 / 02 / 01）

39 - 49

繁體中文

腸膜明串珠菌之葡聚醣蔗糖酶具有將蔗糖中之葡萄糖轉化成葡聚醣之能力。該酵素C端之葡聚醣結合功能區塊（dextran-binding domain，DBD）能與葡聚醣形成可逆性鍵結，具有開發成為親和性標籤之潛力。本研究主要針對腸膜明串珠菌葡聚醣蔗糖酶結構中能與葡聚醣形成可逆性鍵結之最小功能區塊進行探討，以期利用此區塊作為新型親和性標籤。研究中，利用聚合酶連鎖反應進行DBD DNA之選殖與刪減突變，並將不同DNA片段運用於綠螢光蛋白質（green fluorescent protein，GFP）-DBD融合蛋白質表現載體或GFP-删減突變DBD融合蛋白質表現載體之建構。利用電轉形法將表現載體送入大腸桿菌BL21（DE3）中，探討DBD之刪減突變對重組融合蛋白質之表現與純化之影響。結果顯示，DBD-D1為能與葡聚醣形成有效可逆性鍵結之最小功能區塊，且不影響重組融合蛋白質之表現與可溶性。利用DBD-D1作為GFP之親和性標籤可獲得之純化效率高於His tag與DBD。研究結果說明，DBD-D1具有作為新一代親和性標籤之潛力。

Dextransucrase from Leuconostoc mesenteroides is capable of converting glucose in sucrose to dextran. The dextran-binding domain (DBD) on the C-terminal region of dextransucrase forms reversible bonds with dextran and therefore exhibits potential to be developed as an affinity tag. This study mainly explored the minimal DBD of dextransucrase from Leu. mesenteroides to create a novel affinity tag. First, polymerase chain reaction was adopted to perform the cloning and deletion of DBD DNA. Different DNA sequences were used to construct vectors for expressing the green fluorescent protein (GFP)-DBD and GFP-DBD deletion mutants. Electrotransformation was employed to transform the expression vectors into Escherichia coli BL21 (DE3) and determine the effect of DBD deletion mutation on the expression and purification of recombinant fusion proteins. The results revealed that DBD-D1 was the minimal domain involved in effective binding with dextran, and that DBD-D1 fusion did not affect the expression and solubility of the resulting fusion protein. Using DBD-D1 as an affinity tag for GFP enabled greater purification efficiency than did using a His tag and DBD. This indicates that DBD-D1 has the potential to be a next-generation affinity tag.

1. Aldag, C.,Teixeira, D. Nogueira,Leventhal, P. S.(2016).Skin rejuvenation using cosmetic products containing growth factors, cytokines, and matrikines: a review of the literature.Clin. Cosmet. Investig. Dermatol.,9,411-419.
2. Giffard, P. M.,Jacques, N. A.(1994).Definition of a fundamental repeating unit in streptococcal glucosyltransferase glucan-binding regions and related sequences.J. Dent. Res.,73,1133-1141.
3. Gifre, L.,Arís, À.,Bach, A.,Garcia-Fruitós, E.(2017).Trends in recombinant protein use in animal production.Microb. Cell Fact.,16,40.
4. Gupta, S. K.,Shukla, P.(2017).Sophisticated cloning, fermentation, and purification technologies for an enhanced therapeutic protein production: a review.Front. Pharmacol.,8,419.
5. Jozala, A. F.,Geraldes, D. C.,Tundisi, L. L.,Feitosa, V. A.,Breyer, C. A.,Cardoso, S. L.,Mazzola, P. G.,Oliveira-Nascimento, L.,Rangel-Yagui, C. O.,Magalhães, P. O.,Oliveira, M. A.,Pessoa, A., Jr(2016).Biopharmaceuticals from microorganisms: from production to purification.Braz. J. Microbiol.,47,51-63.
6. Kaseda, K.,Kodama, T.,Fukui, K.,Hirose, K.(2001).A novel approach for purification of recombinant proteins using the dextran-binding domain.FEBS Lett.,500,141-144.
7. Kothari, D.,Das, D.,Patel, S.,Goyal, A.(2014).Dextran and Food Application.Polysaccharides,Cham, Switzerland:
8. Krainer, F. W.,Glieder, A.(2015).An updated view on horseradish peroxidases: recombinant production and biotechnological applications.Appl. Microbiol. Biotechnol.,99,1611-1625.
9. Laurino, J. P.,Shi, Q.-W.,Ge, J.(1999).Monoclonal antibodies, antigens and molecular diagnostics: a practical overview.Ann. Clin. Lab. Sci.,29,158-166.
10. Li, X.-J.,Liu, J.-L.,Gao, D.-S.,Wan, W.-Y.,Yang, X.,Li, Y.-T.,Chang, H.-T.,Chen, L.,Wang, C.-Q.,Zhao, J.(2016).Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.Protein Expr. Purif.,119,51-56.
11. Lichty, J. J.,Malecki, J. L.,Agnew, H. D.,Michelson-Horowitz, D. J.,Tan, S.(2005).Comparison of affinity tags for protein purification.Protein Expr. Purif.,41,98-105.
12. Ma, H.,O'Kennedy, R.(2017).Recombinant antibody fragment production.Methods,116,23-33.
13. Meng, X.-F.,Gangoiti, J.,Bai, Y.-X.,Pijning, T.,Van Leeuwen, S. S.,Dijkhuizen, L.(2016).Structure-function relationships of family GH70 glucansucrase and 4,6-α-glucanotransferase enzymes, and their evolutionary relationships with family GH13 enzymes.Cell. Mol. Life Sci.,73,2681-2706.
14. Monchois, V.,Willemot, R. M.,Monsan, P.(1999).Glucansucrases: mechanism of action and structure-function relationships.FEMS Microbiol. Rev.,23,131-151.
15. Morris, J.,Jayanthi, S.,Langston, R.,Daily, A.,Kight, A.,McNabb, D. S.,Henry, R.,Kumar, T. K. S.(2016).Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.Protein Expr. Purif.,126,93-103.
16. Ogez, J. R.,Hodgdon, J. C.,Beal, M. P.,Builder, S. E.(1989).Downstream processing of proteins: recent advances.Biotechnol. Adv.,7,467-488.
17. Olempska-Beer, Z. S.,Merker, R. I.,Ditto, M. D.,DiNovi, M. J.(2006).Food-processing enzymes from recombinant microorganisms—a review.Regul. Toxicol. Pharmacol.,45,144-158.
18. Oliveira, C.,Domingues, L.(2018).Guidelines to reach high-quality purified recombinant proteins.Appl. Microbiol. Biotechnol.,102,81-92.
19. Pina, A. S.,Lowe, C. R.,Roque, A. C.(2014).Challenges and opportunities in the purification of recombinant tagged proteins.Biotechnol. Adv.,32,366-381.
20. Sanchez-Garcia, L.,Martín, L.,Mangues, R.,Ferrer-Miralles, N.,Vázquez, E.,Villaverde, A.(2016).Recombinant pharmaceuticals from microbial cells: a 2015 update.Microb. Cell Fact.,15,33.
21. Semikhin, A. S.,Liashchuk, A. M.,Mezentseva, M. V.,Tregubova, M. I.,Sergienko, O. V.,Poletaeva, N. N.,Narodnotskiĭ, B. S.,Koriagina, A. S.,Lunin, V. G.,Gintsburg, A. L.(2009).Human recombinant interferon-β constructed on the basis of affinity-binding domain technology.Mol. Gen. Mikrobiol. Virusol.,4,38-41.
22. Soto-Rodríguez, J.,Coyle, B. L.,Samuelson, A.,Aravagiri, K.,Baneyx, F.(2017).Affinity purification of Car9-tagged proteins on silica matrices: Optimization of a rapid and inexpensive protein purification technology.Protein Expr. Purif.,135,70-77.
23. Suwannarangsee, S.,Moulis, C.,Potocki-Veronese, G.,Monsan, P.,Remaud-Simeon, M.,Chulalaksananukul, W.(2007).Search for a dextransucrase minimal motif involved in dextran binding.FEBS Lett.,581,4675-4680.
24. Terpe, K.(2003).Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems.Appl. Microbiol. Biotechnol.,60,523-533.
25. van Hijum, S. A.,Kralj, S.,Ozimek, L. K.,Dijkhuizen, L.,van Geel-Schutten, I. G.(2006).Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria.Microbiol. Mol. Biol. Rev.,70,157-176.
26. Waugh, D. S.(2005).Making the most of affinity tags.Trends Biotechnol.,23,316-320.
27. Wingfield, P. T.(2015).Overview of the purification of recombinant proteins.Curr. Protoc. Protein Sci.,80,6.1.1-6.1.35.
28. 陳正文,黃文正,宣詩玲,林俊宏,王志鵬(2016)。新型大腸桿菌/乳酸桿菌穿梭載體之建構與應用。臺灣農業化學與食品科學，54，236-246。
29. 黃文正,曾皓真,方健宇,高俊偉,陳正文,林俊宏,王志鵬(2013)。自發酵蔬果中篩選具耐酸、耐膽鹽及抑菌能力之乳酸菌。台灣食品科學技術學會第四十三次會員大會手冊，臺中，臺灣: