The Achilles Heel of Protein Biochemistry: Insolubility of Recombinant Proteins-A Case Study About Producing a Rice...

Biochemical characterization of proteins is fundamental to understanding their function. Typically, research in protein structure/function requires reasonable quantities of the protein of interest. Because of the low abundances in their natural environment, the heterogenous state of post-translational modifications, and the difficulty of obtaining the tissue containing the protein of interest, recombinant protein production is usually employed. One of the major difficulties impeding advances in biotechnological research is protein insolubility, undermining further downstream research and applications. Escherichia coli strains are popular hosts for protein production but are often unfit for the expression of eukaryotic sequences due to the absence of proper post-translational modifications, some of which are crucial for protein folding and activity. Here, we showcase the challenges researchers may be confronted with when trying to produce proteins recombinantly, by using OsAPSE, an enzyme from rice, as an example of a difficult-to-produce protein. Several production hosts were explored, and best results were obtained when OsAPSE was produced in E. coli combined with a solubility tag or when a higher eukaryotic system was used. This study highlights common pitfalls in protein research and provides strategies to overcome them, making it a case study for researchers facing similar challenges.