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.
Keum
Jung-Won
JW
School of Chemical and Biological Engineering, College of Engineering, Seoul National University, Seoul 151-742, Republic of Korea.
Ahn
Jin-Ho
JH
Choi
Cha-Yong
CY
Lee
Kyung-Ho
KH
Kwon
Yong-Chan
YC
Kim
Dong-Myung
DM
D002457:Cell Extracts / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis; D016385:TATA Box / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*
D002457:Cell Extracts; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins