Computational and Experimental Studies of Divergent Clinical Effects in Proximate Thin Filament Mutations

Abstract

Systemic Lupus Erythematosus is a debilitating autoimmune disease that afflicts about 1.5 million Americans today. Although SLE is a multifactorial disease, SLE can arise with disruptions in two genes: Dnase1 and Dnase1L3. While the structure is known for DNase1, the structure for Dnase1L3 is unknown. To study the structure and function of the Dnase1L3 enzyme, we designed a heterologous expression system and purification protocol for mouse DNase1L3 in E. coli. This system will allow us to characterize the enzymatic activity of the wild-type enzyme. Towards the purpose of solving its 3D structure with X-Ray crystallography we proceeded to the crystallization step of Dnase1L3. A variety of culturing conditions, purification methods, and characterization techniques were performed on purified DNase1L3. After testing a few different methods, we successfully expressed DNase1-L3 in E. coli using Rosetta-gami cells as a fusion protein to Maltose Binding Protein. The fusion enzyme was isolated via amylose affinity resin. After elution from the affinity column, the enzyme was cleaved with TEV protease and further purified on S-resin as a defined peak. Finally, Dnase1L3 was purified using size exclusion chromatography. The activity of purified DNase1-L3 was assessed through digestion of plasmid DNA. The identify of Dnase1L3 was confirmed by Western blot. Now that we have obtained an efficient purification method, verified the activity, and confirmed identity with specific antibodies, we are interrogating the structural aspects of the enzyme through X-Ray crystallography. The structural composition of this enzyme will be invaluable as pertaining to possible use in treating Systemic Lupus Erythematosus.