Pyrrolysyl-tRNA synthetase expression was verified via an added N-terminal Strep-tag. After cell growth and harvesting, cell pellets were dissolved in PBS buffer (58 mM Na2HPO4, 17 mM NaH2PO4, 68 mM NaCl) and ruptured by bead-beating as described earlier (Neves et al., 2010). Strep-PylRS was purified with Strep-Tactin® Resin (IBA). Fractions containing PylRS were selected by SDS-Page and verified by immunoblotting with a 1:100 diluted Strep-Tactin HRP (IBA) conjugate according to manufacturer information. HRP activity was detected using the Amersham ECL Prime Western Blotting Kit (GE Healthcare).