Optimization of expression and purification of recombinant Apollo (SNM1B) protein using affinity chromatography

Abstract

Apollo, also referred to as SNM1B, is a nuclease involved in telomere stability and DNA repair, and its dysfunction is associated with the development of a number of pathologies, including hereditary kidney cancer. The growing interest to this protein is due to its possible use as a target for antitumor therapy. The present study focuses on the optimization of the expression and isolation of the homogenous recombinant Apollo (SNM1B) WT protein by means of affinity chromatography purification. Expression vector constructs pET28c-hSNM1BcoCut and pETHSUL-hSNM1BcoCut were used to produce the protein after their transformation into Escherichia coli (E. coli) Rosetta2 (DE3) and NovaXG strains. Variation in the induction parameters such as concentration of IPTG, incubation temperature, presence of detergent NP-40, glycerol, urea and duration of the post-induction incubations all were subjected to rigorous testing, in order to ascertain the optimal conditions for the obtaining soluble recombinant proteins. The purification process was conducted through the utilization of Ni-NTA affinity chromatography. The purity and solubility of the protein were evaluated by SDS-PAGE. The obtained data allowed to determine effective conditions for producing the soluble and highly homogenous Apollo protein suitable for further studies, such as protein-protein interaction, DNA binding, immunization to obtain specific antibodies, and utilization for screening of inhibitors for cancer therapy.