细菌中功能活跃的人 CT2（SLC22A16）肉碱转运蛋白的过度表达。
Bacterial over-expression of functionally active human CT2 (SLC22A16) carnitine transporter.
机构信息
Department DiBEST Biologia, Ecologia, Scienze Della Terra Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via P. Bucci cubo 4C, 87036, Arcavacata di Rende, Italy.
CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnology IBIOM, via Amendola 122/O, 70126, Bari, Italy.
出版信息
Mol Biol Rep. 2022 Aug;49(8):8185-8193. doi: 10.1007/s11033-022-07491-1. Epub 2022 May 24.
BACKGROUND
Escherichia coli is a widely used tool for the over-expression of human proteins for studying structure and function. The toxicity of human proteins for E. coli often hampers the expression. This study aims to find conditions for the expression of a membrane transporter known as the carnitine transporter CT2. The knowledge on this transporter is scarce, thus obtaining the recombinant protein is very important for further studies.
METHODS AND RESULTS
The cDNAs coding for human CT2 (hCT2) was cloned in the pH6EX3 vector and different transformed E. coli strains were cultured in absence or in presence of glucose. hCT2 expression was obtained. The protein was purified and reconstituted into proteoliposomes in a functionally active state.
CONCLUSIONS
Using the appropriate IPTG concentration, together with the addition of glucose, hCT2 has been expressed in E. coli. The protein is active and shows capacity to transport carnitine in proteoliposomes. The results have a great interest in basic biochemistry of membrane transporters and applications to human health since hCT2 is involved in human pathology.
背景
大肠杆菌是一种广泛用于表达人类蛋白质以研究结构和功能的工具。然而，人类蛋白质对大肠杆菌的毒性常常会阻碍其表达。本研究旨在寻找一种已知的膜转运蛋白——肉碱转运蛋白 CT2 的表达条件。由于对这种转运蛋白的了解甚少，因此获得重组蛋白对于进一步的研究非常重要。
方法和结果
将编码人 CT2（hCT2）的 cDNA 克隆到 pH6EX3 载体中，并在无葡萄糖或有葡萄糖的情况下培养不同转化的大肠杆菌菌株。hCT2 表达成功。纯化后的蛋白质在功能活性状态下被重组成脂质体。
结论
通过使用适当的 IPTG 浓度，以及添加葡萄糖，可以在大肠杆菌中表达 hCT2。该蛋白质是有活性的，并在脂质体中显示出转运肉碱的能力。由于 hCT2 与人的病理有关，因此该结果在膜转运蛋白的基础生物化学和人类健康的应用方面具有重要意义。
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