Optimization strategies for expression and purification of soluble N-terminal domain of human centriolar protein SAS-6 in Escherichia coli

Abstract

Spindle assembly abnormal protein 6 (SAS-6), a highly conserved centriolar protein, constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. Abnormalities in cartwheel assembly lead to chromosomal dysfunctions. The molecular structure of human SAS-6 (HsSAS-6) and cartwheel hub and how they direct centriole symmetry is unknown. No crystal structure of wildtype HsSAS-6 has been reported to date, since soluble recombinant partial/full-length HsSAS-6 expression and purification posed grand challenges. In the present study we have explored optimization of ten different N terminal SAS-6 fusion proteins expression in a variety of E. coli hosts. During optimization we have included some of the most commonly used purification tags: Histidine tag, maltose-binding protein (MBP), small ubiquitin-related modifier (SUMO) tag and modified MBP tag with surface entropy reduction mutations. We demonstrate several levels of tag assisted solubility and stable expression strategies. We find that the MBP tag accompanied by Surface Entropy Reduction mutations (MBP/SER) in a fixed arm approach rescues the folded SAS-6N protein with significantly improved solubility. This expression of HsSAS-6N in E. coli Rosetta DE3 pLysS expression strain gave rise to high protein expression yielding around 6.0–11.5 mg of soluble protein per liter of growth culture.