Scheme 1.

Construction of a polycistronic vector for expression of EF-P alone or its coexpression with YjeA and YjeK for production of unmodified or modified recombinant EF-P. The coding sequences of EF-P, YjeA, and YjeK were first cloned into pET3aTrm vector (29), and each cassette was excised and subcloned into pST39 vector (29), in the order of YjeA, YjeK, and EF-P, at the indicated restriction sites to generate the recombinant vectors listed. Recombinant proteins, His-EF-P1, His-EF-P2, and His-EF-P3, were purified from E. coli harboring the respective recombinant vectors (29). For production of unmodified EF-P, only the EF-P cassette was inserted into pST39. For production of α-lysyl-EF-P, the YjeA cassette was first inserted into pST39, followed by the EF-P cassette. A recombinant vector encoding all three proteins, EF-P, YjeA, and Yjek, was used for generation of β-lysyl-EF-P.