普通烟草NtASAT2的结构预测、克隆表达及功能验证
Structural prediction,cloning expression,and functional verification of NtASAT2 from Nicotiana tabacum L.
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摘要: 为获得具有活性的普通烟草酰基糖酰基转移酶(NtASAT2),采取生物信息学方法对其序列及结构进行分析和预测,并对其基因进行克隆、原核表达与纯化,通过酶催化反应对获得的重组蛋白进行功能验证。结果表明:在NtASAT2二级结构中,α-螺旋及无规则卷曲占比较大,分别为39.04%和41.13%,且NtASAT2的氨基酸序列与粘毛烟草NacASAT2高度相似;在原核细胞BL21(DE3)表达的重组蛋白中,可溶性NtASAT2含量较少,且NtASAT2与镍柱的亲和力较弱,纯化后仅得到少量目标蛋白;在含有底物的酶催化反应体系中,NtASAT2表现出酶活性并催化生成蔗糖二酯。该研究结果可为NtASAT2在酶催化合成蔗糖酯方面的应用提供理论参考。Abstract: To obtain active acylsugar acyltransferase NtASAT2 from Nicotiana tabacum L., bioinformatic methods were employed to analyze and predict its sequence and structure. The NtASAT2 gene was cloned, prokaryotically expressed in Escherichia coli BL21(DE3), and subsequently purified. The function of the recombinant protein was characterized through enzymatic catalysis. The results showed that in the secondary structure of NtASAT2, α-helices and random coils accounted for 39.04% and 41.13% of the total structure, respectively. The amino acid sequence of NtASAT2 was highly similar to that of Nicotiana glutinosa NacASAT2. The solubility of NtASAT2 in the recombinant protein expressed in E.coli BL21(DE3) was low, and NtASAT2 exhibited weak binding affinity for the nickel column. Consequently, only a small amount of the target protein was successfully purified. In enzymatic reaction systems supplemented with substrates, NtASAT2 exhibited enzymatic activity and catalyzed the production of sucrose diesters. These findings provided a theoretical basis for the application of NtASAT2 in the enzymatic catalysis of sucrose ester synthesis.
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Key words:
- Nicotiana tabacum L. /
- acylsugar acyltransferase /
- sucrose ester /
- cloning /
- expression and purification
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