Automated large‐scale purification of a G protein‐coupled receptor for neurotensin

FEBS Letters

Shiloach, Joseph²  White, Jim F¹  Grisshammer, Reinhard¹  Trinh, Loc B² 

[1]Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892-8030, USA

[2]Biotechnology Unit of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA

关键词: Large-scale purification;    G protein-coupled receptor;    Automation;    Detergent;    Neurotensin receptor;    CHAPS;    3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate;    CHS;    cholesteryl hemisuccinate Tris salt;    GPCR;    G protein-coupled receptor;    H10;    decahistidine tag;    LM;    n-dodecyl-β-D-maltoside;    MBP;    E. coli maltose-binding protein;    NTS1–624;    fusion protein MBP-rT43NTR-TrxA-H10 (see Section 2.1);    NTS1–1023;    fusion protein MBP-N10-Tev-rT43NTR-N5G3S-Tev-G3S-TrxA-H10;    NTS1–1233;    fusion protein MBP-N10-Tev-rT43NTR-CH2-N5G3S-G3S-TrxA-H10;    rT43NTR;    N-terminally truncated rat neurotensin type I receptor NTS1;    Tev;    tobacco etch virus;    TrxA;    E. coli thioredoxin;   

DOI  :  10.1016/S0014-5793(04)00195-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Structure determination of integral membrane proteins requires milligram amounts of purified, functional protein on a regular basis. Here, we describe a protocol for the purification of a G protein-coupled neurotensin receptor fusion protein at the 3-mg or 10-mg level using immobilized metal affinity chromatography and a neurotensin column in a fully automated mode. Fermentation at a 200-l scale of Escherichia coli expressing functional receptors provides the material needed to feed into the purification routine. Constructs with tobacco etch virus protease recognition sites at either end of the receptor allow the isolation of neurotensin receptor devoid of its fusion partners. The presented expression and purification procedures are simple and robust, and provide the basis for crystallization experiments of receptors on a routine basis.

【 授权许可】

Unknown