The presence of a common downstream box enables the simultaneous expression of multiple proteins in an E. coli extract.

In our experiments to produce different combinations of recombinant proteins in a cell-free protein synthesis system derived from Escherichia coli, we found that certain pairs of ORFs were not expressed equally. Instead, only a single DNA species was expressed dominantly, while the expression of the others was almost completely repressed. This bias during the co-expression of the DNA pairs was eliminated when an identical downstream box sequence was added to the 5'-ends of the template DNA pairs. By introducing identical nucleotide sequences of the his-tag or the downstream box of chloramphenicol acetyltransferase (CAT-DB) in front of the target genes that were otherwise not expressed compatibly, both of the encoded proteins were produced at similar productivities. Moreover, in the presence of a common downstream box, multiple genes were simultaneously expressed in the same reaction mixture. We expect that the proposed approach will offer a powerful tool for the preparation of unbiased protein libraries, as well as for studying the structure and functions of interacting proteins.