Figure 3.

Flow diagram of the overexpression of site-directed AcK-incorporated proteins in E. coli BL21 (DE3). Taking EcLeuRS as an example, pAcKRS and pET22b(+)-ecleuS were co-transformed in E. coli BL21 (DE3), and engineered pAcKRS were induced by the addition of Ara. Subsequently, IPTG was added to induce the production of EcLeuRS in the presence of NAM, an inhibitor of CobB. With the assistance of pAcKRS, EcLeuRS was translated in full-length form with incorporation of AcK or in truncated form (terminating at the Lys codon mutation site). All other experimental details were as described previously (43).