Expression of an olfactory receptor in Escherichia coli: Purification, reconstitution, and ligand binding
Expression of an olfactory receptor in Escherichia coli: Purification, reconstitution, and ligand binding
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DOI：
10.1021/bi9612069
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发表时间：
1996-12-17
期刊：
影响因子：
2.9
通讯作者：
Breer, H
中科院分区：
文献类型：
--
作者：
Kiefer, H;Krieger, J;Breer, H
嗅觉受体已在细菌细胞中表达为与谷胱甘肽 S-转移酶 (GST) 的融合蛋白。使用缺乏N末端和第一个跨膜区的突变体或使用在第一个细胞内环中携带三个带正电荷的残基的突变体实现了受体蛋白的过表达，产生约10%的细胞蛋白。过表达的融合蛋白在包涵体中积累，并且可以溶解在去污剂中。通过基于 C 端 6-组氨酸标签的金属螯合色谱法对其进行纯化，并在蛋白水解后去除 GST 部分。将纯化的受体重构为脂质囊泡，并通过光亲和标记和色氨酸荧光测量显示气味配体的特异性结合。因此，首次可以大量使用气味受体/配体对进行生物物理和筛选研究。
An olfactory receptor has been expressed in bacterial cells as a fusion protein with glutathione S-transferase (GST). Overexpression of receptor protein yielding about 10% of the cell protein was achieved with mutants lacking the N-terminus and the first transmembrane region or with mutants carrying three positively charged residues in the first intracellular loop. The overexpressed fusion protein accumulated in inclusion bodies and could be solubilized in detergent. It was purified by metal chelation chromatography based on a C-terminal 6-histidine tag, and the GST portion was removed after proteolytic cleavage. The purified receptor was reconstituted into lipid vesicles and specific binding of odor ligands was shown by photoaffinity labeling and tryptophan fluorescence measurements. Thus, for the first time, an odorant receptor/ligand pair becomes available in large amounts for biophysical and screening studies.