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.