Rapid and easy thermodynamic optimization of the 5'-end of mRNA dramatically increases the level of wild type protein expression in Escherichia coli.
Low levels of expression in Escherichia coli are often observed when using wild type proteins. The addition of an N-terminal His-tag to these same proteins dramatically improves the level of expression. We therefore concluded that post-transcriptional regulation and in particular translational regulation are probably influenced by the presence of the tag. The RNAfold program was used to analyze the 5'-end of the encoding mRNA, and more precisely the area encompassing the Shine-Dalgarno region and the initiation codon ATG. We observed that hairpin loops can be formed and that the stability of these loops correlates with the level of protein expression in E. coli. Our recently developed cloning technology by PCR fragment integration allows us to easily and rapidly introduce mutations anywhere within a gene. In our studies, we used this technology to destabilize the predicted hairpin by introducing silent mutations within the first 72 nucleotides of the coding sequence. As a result of the decreased stability of the RNA hairpins, we could significantly increase the level of expression of wild type proteins and without having to rely on the use of tags in E. coli. In addition, our studies allow us to predict whether or not a protein will be expressed without additional engineering of its encoding gene.
Cèbe
Régis
R
Novartis Institutes for BioMedical Research Basel, Discovery Technologies/Protein Structure Unit, WSJ 88.707, CH-4002 Basel, Switzerland.
Geiser
Martin
M
D020121:5' Untranslated Regions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D064173:Anaphase-Promoting Complex-Cyclosome; D001483:Base Sequence; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D005819:Genetic Markers / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics / Q000378:metabolism; D000099157:Sphingosine Kinase
D020121:5' Untranslated Regions; D048868:Adaptor Proteins, Signal Transducing; D064173:Anaphase-Promoting Complex-Cyclosome; D019485:Bone Morphogenetic Proteins; D005819:Genetic Markers; D017853:Phosphotransferases (Alcohol Group Acceptor); D012333:RNA, Messenger; D043743:Ubiquitin-Protein Ligase Complexes; D000099157:Sphingosine Kinase; C422903:SOST protein, human