Editing Error Correction in Gene Synthesis (section)

== Expression and Purification of Aae-MutS ==

E. coli BL21(DE3) (Novagen, Madison, WI, USA) was transformed with plasmid containing mutS gene and cultured at 37 °C in 1.5 L of YT medium [0.8% (w/v) tryptone, 0.5% (w/v) yeast extract and 0.5% (w/v) NaCl] containing 50 μg/mL ampicillin. When the culture density reached 4 × 10 8 cells/mL, isopropyl β- D -thiogalactopyranoside was added to a final concentration of 100 μM to induce expression. The cells were grown at 37 °C for a further 4 h, harvested by centrifugation and lysed by sonication in buffer I (20 mM Tris-HCl and 50 mM NaCl, pH 7.8). The sonicate was heated to 70 °C for 10 min, centrifuged at 48,000× g for 20 min, and the supernatant was then loaded onto a Talon resin column (40 mL; Clontech, Palo Alto, CA, USA) equilibrated with buffer I. The column was washed with 300 mL of buffer I and then eluted with buffer I containing 150 mM imidazole. The fractions containing AaeMutS were detected by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) and collected, and (NH 4 ) 2 SO 4 was added to the fraction to a final concentration of 1 M. The solution was loaded onto a Toyopearl-Phenyl column (Tosoh, Tokyo, Japan) equilibrated with buffer I containing 1 M (NH4)2SO4 . The column was washed with 100 mL of buffer I containing 1 M (NH 4 ) 2 SO 4 and then eluted with a 300 mL gradient of 1 to 0 M (NH 4 ) 2 SO 4 in buffer I. The fractions containing AaeMutS were detected by SDS-PAGE and concentrated with a Vivaspin concentrator (Vivascience, Göttingen, Germany). The protein solution was dialyzed against 20 mM Tris-HCl and 50 mM NaCl (pH 7.8).