Enhancing Expression of Hydrophobic Peptides in E. coli Via Fusion to Novel Proteins

Abstract

Combinatorial design of libraries of novel proteins is an important method for exploring the sequence space un-tested by nature. Using this strategy, novel proteins that fold into four-helix bundles have been created and characterized. While some library members have been assigned functionality, this is not the case for many of the stable four-helix bundles identified. For example, although S824, a member of the Hecht lab’s second generation library, has been well-characterized as a stable and soluble protein and its four-helix bundle structure has been solved, no function of this protein has been elucidated. This study investigates the potential function of S824 as an expression-enhancing fusion partner for poorly expressed protein products in E. coli. Previously, it has been shown that linking natural proteins that are structurally stable and soluble to a poorly expressed product can greatly enhance the product’s recombinant expression. To determine the if synthetic proteins can also facilitate production in this manner, the ability of S824 to increase expression levels of two difficult-to-express peptides was studied. It was found that S824 allows for recombinant production of LS3, a synthetic membrane-associating peptide, and amyloid beta (1-42), the aggregation prone peptide that forms neurotoxic plaques in Alzheimer’s disease. Further, it was found that fusion to S824 enhances the expression of these peptides beyond levels achieved through expression as part of a construct with the natural protein small ubiquitin-like modifier. Additionally, after optimization of the cleavage of Aβ from S824 using TEV protease and subsequent purification of Aβ, it was determined that 22.7 mg of Aβ can be produced per liter of cell culture using this method, compared to 13 mg reportedly produced by fusion to SUMO. Thus, a more descriptive name for S824 is, in fact, de novo expression enhancing protein (DEEP). The success of DEEP indicates a new application of synthetic proteins to biotechnology.