Xiao Shuyan, Zhao Xiaolin, Finkielstein Carla V, Capelluto Daniel G S
Protein Signaling Domains Laboratory, Department of Biological Sciences, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, 24061, USA.
J Pept Sci. 2014 Mar;20(3):216-22. doi: 10.1002/psc.2604. Epub 2014 Jan 27.
A procedure for obtaining isotopically labeled peptides, by combining affinity chromatography, urea-equilibrated gel filtration, and hydrophobic chromatography procedures, is presented using the Disabled-2 (Dab2) sulfatide-binding motif (SBM) as a proof of concept. The protocol is designed to isolate unstructured, membrane-binding, recombinant peptides that co-purify with bacterial proteins (e.g., chaperones). Dab2 SBM is overexpressed in bacteria as an isotopically labeled glutathione S-transferase (GST) fusion protein using minimal media containing [¹⁵N] ammonium chloride as the nitrogen source. The fusion protein is purified using glutathione beads, and Dab2 SBM is released from GST using a specific protease. It is then dried, resuspended in urea to release the bound bacterial protein, and subjected to urea-equilibrated gel filtration. Urea and buffer reagents are removed using an octadecyl column. The peptide is eluted with acetonitrile, dried, and stored at -80 °C. Purification of Dab2 SBM can be accomplished in 6 days with a yield of ~2 mg/l of culture. The properties of Dab2 SBM can be studied in the presence of detergents using NMR spectroscopy. Although this method also allows for the purification of unlabeled peptides that co-purify with bacterial proteins, the procedure is more relevant to isotopically labeled peptides, thus alleviating the cost of peptide production.
本文介绍了一种通过结合亲和色谱、尿素平衡凝胶过滤和疏水色谱法来获得同位素标记肽段的方法，该方法以Disabled-2（Dab2）硫酸脑苷脂结合基序（SBM）作为概念验证。该方案旨在分离与细菌蛋白（如伴侣蛋白）共纯化的无结构、膜结合的重组肽段。Dab2 SBM在细菌中作为同位素标记的谷胱甘肽S-转移酶（GST）融合蛋白过表达，使用含有[¹⁵N]氯化铵作为氮源的基本培养基。融合蛋白用谷胱甘肽珠纯化，Dab2 SBM使用特定蛋白酶从GST中释放出来。然后将其干燥，重悬于尿素中以释放结合的细菌蛋白，并进行尿素平衡凝胶过滤。使用十八烷基柱去除尿素和缓冲试剂。肽段用乙腈洗脱，干燥后储存在-80°C。Dab2 SBM的纯化可在6天内完成，产量约为2 mg/l培养物。Dab2 SBM的性质可以在去污剂存在的情况下使用核磁共振光谱进行研究。虽然该方法也允许纯化与细菌蛋白共纯化的未标记肽段，但该过程与同位素标记肽段更相关，从而降低了肽段生产的成本。
