Ribosome-binding site interference caused by Shine-Dalgarno-like nucleotide sequences in Escherichia coli cells.
Two-cistronic expression plasmids are useful for high-level expression of heterologous genes in Escherichia coli cells by preventing the inhibition of translational initiation. In the process of constructing a two-cistronic expression plasmid pCbSTCR-4 containing the fragments of the porcine cytochrome b(5) (Psb5) and NADPH-cytochrome P450 reductase (PsCPR) genes as the first and second cistrons, respectively, the presence of a specific region in the first cistron that lowered the accumulation level of the PsCPR was suggested [Kimura, S., et al. (2005) J. Biochem. 137, 523-533]. In this study, a disturbing nucleotide sequence similar to a Shine-Dalgarno (SD) sequence (SD-like sequence), AGGAG, was identified at the 5'-upstream region near the SD sequence for the second cistron. Silent mutations in the SD-like sequence that lowered the similarity to a typical SD sequence increased the accumulation level of PsCPR. SD-like sequences introduced into mono-cistronic expression plasmids for the Psb5 and PsCPR genes also decreased the accumulation level of these proteins. The SD-like sequence also decreased the accumulation level of the insoluble PsCPR protein. This type of ribosome-binding site interference is useful not only for precise control of protein accumulation but also for increasing the soluble form of recombinant proteins in E. coli cells.
Nishizawa
Akito
A
VBL, Hitachi, 316-8511, Japan.
Nakayama
Miki
M
Uemura
Takuya
T
Fukuda
Yoshiyuki
Y
Kimura
Shigenobu
S
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015786:Cytochromes b5 / Q000096:biosynthesis / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012270:Ribosomes / Q000378:metabolism*; D013552:Swine
D011994:Recombinant Proteins; D004247:DNA; D015786:Cytochromes b5; D009251:NADPH-Ferrihemoprotein Reductase