Fermentation Facility, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Department of Applied Science, William & Mary, Williamsburg, VA, USA.
Protein Expr Purif. 2023 Dec;212:106354. doi: 10.1016/j.pep.2023.106354. Epub 2023 Aug 18.
The production of highly purified native soluble proteins in large quantities is crucial for studying protein structure and function. Odorant binding proteins (OBPs) are small, soluble, extracellular proteins with multiple disulfide bonds, whose functions include, but are not limited to, binding hydrophobic molecules and delivering them to their corresponding receptors expressed on insect olfactory receptor neurons. Expression of proteins with multiple disulfide bonds like OBPs usually results in insolubility and low yield, which has been a significant barrier to understanding their biological roles and physiological functions. In the E. coli system, expression of OBPs often results in insoluble inclusion bodies or a limited amount of periplasmic soluble proteins. Although expression of OBPs in eukaryotic systems such as Sf9 insect cells or yeast Pichia pastoris can increase the solubility of the protein, the process remains insufficient. Additionally, monitoring the purity and native apo state of the protein is critical for establishing the correct conformation of the protein. In this study, we employed an E. coli host with an altered intracellular environment to produce cytosolic soluble OBP44a protein, which yielded over 100 mg/L. We monitored the integrity of disulfide bonds throughout the purification process using LC-MS and used NMR to ensure the final product adopted a single conformation. Our study presents an efficient method for obtaining large quantities of soluble proteins in a single conformation, which enables extensive in vitro studies of secreted proteins like OBPs.
大量生产高度纯化的天然可溶性蛋白质对于研究蛋白质结构和功能至关重要。气味结合蛋白（OBP）是具有多个二硫键的小分子量可溶性细胞外蛋白，其功能包括但不限于结合疏水分子并将其递送至表达于昆虫嗅觉受体神经元上的相应受体。具有多个二硫键的蛋白质的表达通常会导致不溶性和低产量，这一直是理解其生物学作用和生理功能的重大障碍。在大肠杆菌系统中，OBP 的表达通常会导致不溶性包涵体或有限量的周质可溶性蛋白。尽管在 Sf9 昆虫细胞或酵母毕赤酵母等真核系统中表达 OBP 可以增加蛋白质的可溶性，但该过程仍然不足。此外，监测蛋白质的纯度和天然 apo 状态对于建立蛋白质的正确构象至关重要。在本研究中，我们使用经过改造的大肠杆菌宿主产生细胞溶质可溶性 OBP44a 蛋白，产量超过 100mg/L。我们在整个纯化过程中使用 LC-MS 监测二硫键的完整性，并使用 NMR 确保最终产物采用单一构象。我们的研究提出了一种获得大量单一构象可溶性蛋白质的有效方法，这使得对 OBPs 等分泌蛋白进行广泛的体外研究成为可能。
