Cloning, Expression, and Purification of zmARG

The ZMO0432 gene encoding zmARG (Met1∼Lys290) was amplified from Z. mobilis subsp. mobilis ZM4 genomic DNA by polymerase chain reaction (PCR) using the two primers of 5′-CGATACCATATGAGTAGTATTAATAAACCGTTGAGACTC ATTTTCCCG-3′ and 5′-CGTCTCGAGTTATTTCCCGATTAA AGGCAGCTCTTCGAG-3′, which contains the NdeI and XhoI restriction sites (underlined), respectively. The amplified PCR product was treated with the restriction enzymes NcoI (New England Biolabs, Beverley, MA, United States) and XhoI (New England Biolabs, Beverley, MA, United States) and was inserted into the pSKB3 bacterial expression vector that expresses 25 extra residues at the N-terminus including a cleavable six-histidine residues followed by the tobacco etch virus (TEV) protease cleavage site. The constructed recombinant plasmid was transformed into Escherichia coli BL21^∗(DE3) Star that was grown in LB medium or in a seleno-L-methionine (Se-Met) based medium in B834 (DE3) containing 100 μg⋅ml^–1 ampicillin at 310 K. When the optical density at 600 nm reached 0.5, the fusion protein was expressed by adding 1.0 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) into the culture media followed by incubation for an additional 8 h at 310 K. The culture was harvested by centrifugation at 5,000 g at 277 K. The cell pellet was resuspended in an ice-cold buffer A (20 mM Tris–HCl (pH 7.5) and 150 mM NaCl) and disrupted by ultrasonication. The cell debris was removed by centrifugation at 11,000 g for 1 h. The expressed zmARG fusion protein was initially bound by a 5 ml HisTrapHP chelating column (GE Healthcare, Uppsala, Sweden) and the bound protein was eluted by a 500 mM imidazole gradient in buffer A. The eluted zmARG protein was incubated with the recombinant TEV protease and simultaneously dialyzed to remove the salt. After dialysis, the added TEV protein was removed by reloading the protein on the 5 ml HisTrap HP chelating column. The protein containing two additional amino acids (GH) at the N-terminus was further purified by gel filtration on a Superdex 200 column (GE Healthcare, Uppsala, Sweden) under the same buffer condition. For phasing, Se-Met substituted zmARG was prepared in a similar manner to the native protein.