文章詳目資料

臺灣農業化學與食品科學 Scopus

  • 加入收藏
  • 下載文章
篇名 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
卷期 57:1
並列篇名 Application of the Minimal Dextran–Binding Domain as an Affinity Tag for Recombinant Protein Purification
作者 黃文正陳正文卓侑霖林俊宏宣詩玲王志鵬
頁次 039-049
關鍵字 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 mesenteroidesdextransucrasedextran-binding domainaffinity tagScopusTSCI
出刊日期 201902
DOI 10.6578/TJACFS.201902_57(1).0005

中文摘要

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DBDDNA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-刪減突變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。

英文摘要

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.

相關文獻