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毕赤酵母中功能性人源葡萄糖转运蛋白 1 的表达和功能表征的结构生物学工作流程。

Structural biology workflow for the expression and characterization of functional human sodium glucose transporter type 1 in Pichia pastoris.

机构信息

Biophysics Unit, Department of Biochemistry and Molecular Biology, School of Medicine, Universitat Autònoma de Barcelona, 08193, Cerdanyola del Vallés, Catalonia, Spain.

Department of Biochemistry and Biophysics, Stockholm University, SE-10691, Stockholm, Sweden.

出版信息

Sci Rep. 2019 Feb 4;9(1):1203. doi: 10.1038/s41598-018-37445-2.

DOI:10.1038/s41598-018-37445-2
PMID:30718602
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6362292/
Abstract

Heterologous expression of human membrane proteins is a challenge in structural biology towards drug discovery. Here we report a complete expression and purification process of a functional human sodium/D-glucose co-transporter 1 (hSGLT1) in Pichia pastoris as representative example of a useful strategy for any human membrane protein. hSGLT1 gene was cloned in two different plasmids to develop parallel strategies: one which includes green fluorescent protein fusion for screening optimal conditions, and another for large scale protein production for structural biology and biophysics studies. Our strategy yields at least 1 mg of monodisperse purified recombinant hSGLT1 per liter of culture, which can be characterized by circular dichroism and infrared spectroscopy as an alpha-helical fold protein. This purified hSGLT1 transports co-substrates (Na and glucose) and it is inhibited by phlorizin in electrophysiological experiments performed in planar lipid membranes.

摘要

人源膜蛋白的异源表达是药物发现的结构生物学领域的一项挑战。在此，我们报告了一个功能正常的人源钠/葡萄糖协同转运蛋白 1（hSGLT1）在巴斯德毕赤酵母中的完整表达和纯化过程，这是针对任何人类膜蛋白的一种有用策略的代表性示例。hSGLT1 基因被克隆到两个不同的质粒中，以开发并行策略：一个包含绿色荧光蛋白融合，用于筛选最佳条件，另一个用于大规模蛋白质生产，用于结构生物学和生物物理学研究。我们的策略至少可以从每升培养物中获得 1 毫克单分散纯化的重组 hSGLT1，其可以通过圆二色性和红外光谱来表征为具有α-螺旋折叠结构的蛋白。这种纯化的 hSGLT1 可以转运共底物（Na 和葡萄糖），并且在平面脂质膜中进行的电生理学实验中，它可以被根皮苷抑制。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/5c6303da025d/41598_2018_37445_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/e2dd1716ccc8/41598_2018_37445_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/08e12deb566e/41598_2018_37445_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/e4113429a318/41598_2018_37445_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/eaa2077cb795/41598_2018_37445_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/0206c0fae138/41598_2018_37445_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/bb887fbb9714/41598_2018_37445_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/5c6303da025d/41598_2018_37445_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/e2dd1716ccc8/41598_2018_37445_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/08e12deb566e/41598_2018_37445_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/e4113429a318/41598_2018_37445_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/eaa2077cb795/41598_2018_37445_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/0206c0fae138/41598_2018_37445_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/bb887fbb9714/41598_2018_37445_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5ae/6362292/5c6303da025d/41598_2018_37445_Fig7_HTML.jpg

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