The translation of recombinant proteins in E. coli can be improved by in silico generating and screening random libraries of a -70/+96 mRNA region with respect to the translation initiation codon.
Recombinant protein translation in Escherichia coli may be limited by stable (i.e. low free energy) secondary structures in the mRNA translation initiation region. To circumvent this issue, we have set-up a computer tool called 'ExEnSo' (Expression Enhancer Software) that generates a random library of 8192 sequences, calculates the free energy of secondary structures of each sequence in the -70/+96 region (base 1 is the translation initiation codon), and then selects the sequence having the highest free energy. The software uses this 'optimized' sequence to create a 5' primer that can be used in PCR experiments to amplify the coding sequence of interest prior to sub-cloning into a prokaryotic expression vector. In this article, we report how ExEnSo was set-up and the results obtained with nine coding sequences with low expression levels in E. coli. The free energy of the -70/+96 region of all these coding sequences was increased compared to the non-optimized sequences. Moreover, the protein expression of eight out of nine of these coding sequences was increased in E. coli, indicating a good correlation between in silico and in vivo results. ExEnSo is available as a free online tool.
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D020121:5' Untranslated Regions / Q000737:chemistry; D000225:Adenine / Q000737:chemistry; D018387:Codon, Initiator; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D016296:Mutagenesis; D010442:Peptide Chain Initiation, Translational; D012333:RNA, Messenger / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017423:Sequence Analysis, RNA; D012984:Software
D020121:5' Untranslated Regions; D018387:Codon, Initiator; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000225:Adenine