食品科学 ›› 2022, Vol. 43 ›› Issue (12): 187-188.doi: 10.7506/spkx1002-6630-20210620-237

• 生物工程 • 上一篇    

人乙醛脱氢酶2与NusA的融合表达

缪士涛,胡敏,宫兴文   

  1. (浙江工商大学食品与生物工程学院,浙江 杭州 310018)
  • 发布日期:2022-07-01
  • 基金资助:
    浙江省自然科学基金项目(LY18C200004)

Fusion Expression of Human Acetaldehyde Dehydrogenase 2 with NusA

MIAO Shitao, HU Min, GONG Xingwen   

  1. (College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China)
  • Published:2022-07-01

摘要: 通过将乙醛脱氢酶2(acetaldehyde dehydrogenase 2,ALDH2)与NusA-tag融合表达,以获得能够在大肠杆菌中可溶性表达并且具有较好活性的重组蛋白。首先,根据Aldh2的基因序列设计引物,引入EcoRI和XhoI的酶切位点,聚合酶链式反应扩增出Aldh2基因片段,连接到pMD19-T-simple载体,并转化到大肠杆菌DH5α菌株。测序正确后,双酶切处理,将Aldh2基因片段克隆到表达载体pET44b(+)上NusA-tag下游的EcoRI和XhoI酶切位点之间,得到pET44b(+)-NusA-Aldh2重组载体,转化入大肠杆菌BL21(DE3)菌株。经异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)诱导,表达的融合蛋白具有良好的溶解性,主要存在于上清液中。融合蛋白的最优表达条件为IPTG浓度0.25 mmol/L、诱导温度37 ℃、诱导时间3 h。重组蛋白的最适反应温度为37 ℃,在pH 7.0时达到最好的催化效果。不同金属离子如Ca2+、K+、Na+、Mg2+、Mn2+都对酶有激活作用,且Mg2+效果最好,最佳的酶活性为1.64 U/mL。而野生型ALDH2在大肠杆菌中表达时完全以无活性的包涵体形式存在,复性后得到的酶活性为1.43 U/mL。本研究通过引入NusA进行融合表达,实现了ALDH2在大肠杆菌中的可溶性表达,且重组蛋白的活性优于包涵体复性蛋白。这些结果表明利用NusA进行融合表达是制备重组ALDH2的一个良好方法。

关键词: 乙醛脱氢酶2;包涵体;融合表达;NusA-tag;重组蛋白

Abstract: Aldehyde dehydrogenase 2 (ALDH2) was fused with NusA-tag to solubly express a recombinant protein with good activity in Escherichia coli. Primers were designed according to the gene sequence of Aldh2, and the restriction enzyme sites EcoRI and XhoI were inserted into the 5’ end to amplify the Aldh2 gene fragment, which was then ligated into the pMD19-T-Simple vector and transformed into E. coli DH5α. After sequencing, the correct Aldh2 gene fragment was cloned into the expression vector pET44b(+) at the downstream of NusA-tag between EcoRI and XhoI and transformed into E. coli BL21(DE3). Protein expression was induced using isopropyl-β-D-thiogalactopyranoside (IPTG), and the resultant fusion protein had good solubility and mainly existed in the supernatant. The optimal expression conditions were found to be induction at 37 ℃ for 3 h with 0.25 mmol/L IPTG. The optimal reaction pH and temperature for the recombinant protein were 7.0 and 37 ℃, respectively. The metal ions Ca2+, K+, Na+, Mg2+ and Mn2+ could improve the enzyme activity, and Mg2+ had the most pronounced effect, increasing the activity to 1.64 U/mL. The wild-type ALDH2 was expressed as an inclusion body in E. coli with an activity of 1.43 U/mL. These results indicated that the fusion expression with NusA is a good method for the preparation of recombinant ALDH2 in E. coli.

Key words: aldehyde dehydrogenase 2; inclusion body; soluble expression; NusA-tag; recombinant protein

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