Tuning protein expression using synonymous codon libraries targeted to the 5' mRNA coding region.
In bacteria, the 5' mRNA coding region plays an important role in determining translation output. Here, we report synthetic sequences that when placed in the 5'-mRNA coding region, leading to recombinant proteins containing short N-terminal extensions, virtually abolish, enhance or produce intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding sequence allowed tuning of protein expression over ~300-fold with preservation of amino acid identity. This approach is simple and should be generally applicable in bacteria. The data support that features in the 5' mRNA coding region near the AUG start codon are key in determining translation output and hence is important to recombinant and, most certainly, endogenous gene expression.
Department of Cellular and Molecular Medicine, The Panum Institute, University of Copenhagen, Blegdamsvej 3b, 2200 Copenhagen, Denmark.
Lise
L
Goltermann
Martin
M
Borch Jensen
Thomas
T
Bentin
D020121:5' Untranslated Regions; D001483:Base Sequence; D003062:Codon; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics
D020121:5' Untranslated Regions; D003062:Codon; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins