Abstract
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.
