Conclusions
In vivo residue-specific incorporation of Dopa in AY tyrosine-auxotrophic E. coli cells resulted in 4.19 mg/L of purified Dopa-MAP with over 90% Dopa incorporation yield. In order to enhance the production yield of Dopa-MAP, coexpression of two Dopa-recognizing TyrRSs (MjTyrRS-Dopa and EcTyrRS) was performed. Coexpression of MjTyrRS as well as MjtRNATyrAUA increased the production yield of Dopa-MAP by 57%. Coexpression of EcTyrRS resulted in a 72% enhanced production yield of Dopa-MAP. Reactivities of Dopa in the Dopa-MAP samples were confirmed by NBT staining of protein gels. MALDI-TOF MS analysis showed that all Dopa-MAP samples have over 90% Dopa incorporation yield. Although the increase in the production yield of Dopa-MAP was moderate in this study, application of these strategies to other MAP proteins with different Dopa contents, especially those with lower Dopa contents, may result in a greater increase in production yield.