FIG. 3.

Purification of recombinant MBP-nsp13 and MBP-nsp13_KA fusion proteins from E. coli cells. (A) Aliquots taken at each step of the purification protocol were analyzed on a sodium dodecyl sulfate-12% polyacrylamide gel, and the proteins were stained with Coomassie brilliant blue dye. Lanes: 1, protein molecular mass markers, with masses indicated on the left (in kilodaltons); 2, cleared lysate of IPTG-induced E. coli TB1 bacteria transformed with the expression plasmid pMal-SARS-CoV-nsp13; 3 and 4, peak fractions 1 and 2, respectively, from the amylose-agarose chromatography column; 5, pooled peak fractions from the Superdex 200 column; 6, cleared lysate of IPTG-induced E. coli TB1 bacteria transformed with the expression plasmid pMal-SARS-CoV-nsp13_KA; 7 and 8, peak fractions 1 and 2, respectively, from the amylose-agarose chromatography column; 9, pooled peak fractions from the Superdex 200 column. The fusion proteins are indicated by an arrowhead. (B) Western immunoblot analysis with SARS-CoV nsp13-specific rabbit antiserum. Lanes: 1, cleared lysate of E. coli TB1 bacteria transformed with the expression plasmid pMal-SARS-CoV-nsp13; 2, cleared lysate of IPTG-induced E. coli TB1 bacteria transformed with the expression plasmid pMal-SARS-CoV-nsp13; 3, purified MBP-nsp13; 4, purified MBP-nsp13_KA. The positions of protein molecular mass markers are indicated on the left (in kilodaltons).