title: 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.
abstract: 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.
authors: AFMB UMR6098 CNRS/Université Aix-Marseille I & II, Case 932, 163 Avenue de Luminy, 13288 Marseille Cedex 09, France.
authors: S
authors: S
authors: Care
authors: C
authors: C
authors: Bignon
authors: M C
authors: MC
authors: Pelissier
authors: E
authors: E
authors: Blanc
authors: B
authors: B
authors: Canard
authors: B
authors: B
authors: Coutard
mesh_terms: 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
chemical_list: D020121:5' Untranslated Regions; D018387:Codon, Initiator; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000225:Adenine