E coli bl21 codonplus de3

An expression vector (pET-#0219) that contains the cDNA fragment encoding amino acids between 45 and 307 of the wild-type AtOR (At5g61670) protein from Arabidopsis thaliana was amplified by RT-PCR using primers, P217 and P227 (Figure 1). The amplified DNA fragments were ligated with the expression vector pET24a (+) digested with NdeI and XhoI using In-Fusion HD cloning kit (Takara bio, Japan). cDNA fragments encoding amino acids between 45 and 95, and between 220 and 307 of AtOR were amplified by RT-PCR using primer sets of P227 and P229, and of P228 and P217, respectively. These DNA fragments were ligated with the expression vector pET24a(+) digested with NdeI and XhoI using In-Fusion HD cloning kit, designated pET-#0220. The amplified DNA fragments using primers P206 and P217 were ligated with pET24a (+), designated pET-#0213. The sequences of the DNA fragments were confirmed. The E. coli BL21-CodonPlus (DE3; Agilent Technologies, U.S.A.) was used to express the OR fusion proteins with 6× His tags. The expression and purification of these proteins were performed as described.^{9 (link)}Figure 1.

Schematic presentation of AtOR (At5g61670) protein and variants. The positions of AtOR truncations are indicated. TP, transit peptide; TM: transmembrane domain; Zn-finger: zinc finger motif. Arrows show the position of the primers used in the PCR reactions.