Heterologous expression and purification of recombinant proteins

The M. globosa CYP51 gene (Mglob51 - KEGG M. globosa genome database http://www.genome.jp/dbget-bin/www_bget?mgl:MGL_2415) and CYP5218 gene (Mglob5218 - http://www.genome.jp/dbget-bin/www_bget?mgl:MGL_3996) were synthesized by Eurofins MWG Operon (Ebersberg, Germany) and cloned into the pCWori⁺ expression vector. The first eight amino acids of each protein were changed to ‘MALLLAVF’⁴⁴ and a four-histidine tag added to the C’ terminus. The pCWori⁺:Mglob51 and pCWori⁺:Mglob5218 constructs were transformed into DH5α E. coli cells. M. globosa CYP51 was expressed as previously described³¹ whilst for CYP5218 the expression temperature was lowered to 20 °C for 18 h at 180 rpm. Recombinant proteins were isolated according to the method of Arase et al.⁴⁵ and solubilized CYP51 and CYP5218 proteins were purified using Ni²⁺-NTA agarose²⁸. Protein purity was assessed by SDS polyacrylamide gel electrophoresis.