The presence of a common downstream box enables the simultaneous expression of multiple proteins in an E. coli extract |
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Authors: | Keum Jung-Won Ahn Jin-Ho Choi Cha-Yong Lee Kyung-Ho Kwon Yong-Chan Kim Dong-Myung |
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Affiliation: | School of Chemical and Biological Engineering, College of Engineering, Seoul National University, Seoul 151-742, Republic of Korea. |
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Abstract: | 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. |
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Keywords: | Cell-free protein synthesis S30 extract Co-expression Downstream box |
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