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

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.

Hypomorphic mutations in human DNA ligase IV lead to compromised DNA binding efficiency, hydrophobicity and thermal stability.

Studies have shown that Lig4 syndrome mutations in DNA ligase IV (LigIV) are compromised in its function with residual level of double strand break ligation activity in vivo. It was speculated that Lig4 syndrome mutations adversely affect protein folding and stability. Though there are crystal structures of LigIV, there are no reports of crystal structures of Lig4 syndrome mutants and their biophysical characterization to date. Here, we have examined the conformational states, thermal stability, hydrophobicity and DNA binding efficiency of human DNA LigIV wild type and its hypomorphic mutants by far-UV circular dichroism, tyrosine and tryptophan fluorescence, and 1-anilino-8-naphthalene-sulfonate binding, dynamic light scattering, size exclusion chromatography, multi-angle light scattering and electrophoretic mobility shift assay. We show here that LigIV hypomorphic mutants have reduced DNA-binding efficiency, a shift in secondary structure content from the helical to random coil, marginal reduction in their thermal stability and increased hydrophobicity as compared to the wild-type LigIV.