融合表达氨基转移酶DsaD和异构酶DsaE合成L-别异亮氨酸

Overexpression of the aminotransferase DsaD and isomerase DsaE as a fusion protein to synthesize L-allo-isoleucine

摘要:

【背景】L-异亮氨酸(L-isoleucine，L-Ile)和L-别异亮氨酸(L-allo-isoleucine，L-allo-Ile)是自然界中广泛存在的一对同分异构体。抗感染抗生素desotamides结构中含L-别异亮氨酸结构单元，其生物合成途径中的氨基转移酶DsaD和异构酶DsaE可以协作催化L-异亮氨酸和L-别异亮氨酸相互转化。【目的】通过理性设计，使氨基转移酶DsaD和异构酶DsaE融合表达，研究融合蛋白DsaDE催化异亮氨酸和别异亮氨酸相互转化的功能。【方法】利用PCR分别扩增dsaE基因编码区DNA片段、以及含dsaD基因编码区和114个碱基接头序列的DNA片段dsaD-linker，利用酶切位点Kpn I将dsaE和dsaD-linker相连，形成dasDE重组序列，并克隆至pET28a(+)中，将重组质粒pET28a-dsaDE转化至Escherichia coli BL21(DE3)中进行融合表达，利用Ni-NTA亲和层析法纯化融合蛋白DsaDE；分别以L-异亮氨酸和L-别异亮氨酸为底物进行融合蛋白的体外酶活性检测，利用高效液相色谱对酶反应产物进行分析。【结果】PCR验证、酶切验证以及测序结果证明pET28a-dsaDE重组载体具有正确序列；N-末端和C-末端融合6个组氨酸标签的融合蛋白DsaDE在E. coli BL21(DE3)中获得可溶性表达，经Ni-NTA亲和层析法一步纯化获得纯度约95%的融合蛋白，纯化的融合蛋白DsaDE具有较好的活性，能够催化L-isoleucine和L-allo-isoleucine间的相互转化。【结论】氨基转移酶DsaD和异构酶DsaE成功融合表达，经一步Ni-NTA亲和层析法纯化即可获得纯度较高的融合蛋白，融合蛋白同时具有氨基转移酶和异构酶的活性，为进一步研究L-别异亮氨酸的工业化生产奠定了基础。

关键词:氨基转移酶，异构酶，融合蛋白，异亮氨酸

Abstract:

[Background] L-isoleucine (L-Ile) and L-allo-isoleucine (L-allo-Ile) are two isomers in nature. The anti-infective antibiotic desotamides contain an L-allo-Ile moiety, and the aminotransferase DsaD and isomerase DsaE in their biosynthetic pathway catalyze the interconversion between L-Ile and L-allo-Ile. [Objective] To overexpress the aminotransferase DsaD and isomerase DsaE as a fusion protein DsaDE, and assay its activity to catalyze the interconversion between L-Ile and L-allo-Ile. [Methods] The coding region of dsaE and dsaD-linker (coding region of dasD containing 114 bp linker) were amplified by PCR; the purified DNA fragments were digested and ligated into pET28a(+), and the resulting recombinant plasmid was transformed into Escherichia coli BL21(DE3) to overexpress the fusion protein; the fusion protein DsaDE was purified by Ni-NTA affinity chromatography, then the activity of the fusion protein DsaDE was assayed using L-Ile or L-allo-Ile as substrate, respectively; the reaction mixtures were analyzed by high performance liquid chromatography (HPLC). [Results] PCR analyses, restriction endonuclease digestion and sequencing demonstrated that dasE and dsaD-linker were correctly ligated into pET28a(+); the fusion protein DsaDE with His6 tags at both the N-terminus and C-terminus was solubly produced in E. coli BL21(DE3) and purified to ~95% homogeneity. The purified fusion protein can catalyze the interconversion between L-Ile and L-allo-Ile efficiently in vitro. [Conclusion] The aminotransferase DsaD and isomerase DsaE were successfully overexpressed as a fusion protein; the fusion protein was readily purified by one-step Ni-NTA affinity chromatography; and purified fusion protein can catalyze the interconversion between L-Ile and L-allo-Ile efficiently, laying the foundation for further research on the industrial production of L-allo-Ile.

Key words:Aminotransferase, Isomerase, Fusion protein, Isoleucine