@article{Kües_Kia_Lakkireddy_Zomorrodi_2021, title={Transcriptional and translational leakiness in expression of a fungal cyclopropane fatty acid synthase in the Escherichia coli pET-vector expression system}, volume={15}, url={https://abap.co.in/index.php/home/article/view/15}, DOI={10.5530/ctbp.2021.1.1}, abstractNote={<p style="text-align: justify;">We expressed the putative cyclopropane synthase <em>Cfs1</em> from the mushroom <em>Coprinopsis cinerea</em> and the native bacterial Cfa enzyme in <em>Escherichia coli</em> as fusion proteins with an Nterminal 10xHis-tag and a proteolytic Factor Xa site, using the commercial pET-16b vector with the T7lac promoter for expression. Proteomics detected both proteins with high Mascot scores via trypsin-digested peptides in total cytoplasmic protein samples from transformed BL21(DE3) bacteria, induced by IPTG (isopropyl β-D-1- thiogalactopyranoside) for T7 RNA polymerase production from theT7 phage gene 1 present in the host genome under control by the <em>lacUV5</em> promoter. In lower amounts, <em>Cfs1</em> and Cfa were also found in protein samples from non-induced cells based on the known transcriptional leakiness of the pET-system in expression of T7 RNA polymerase. As a novelty, the results on <em>Cfs1</em> from proteomics suggest that the sequence at the pET-16b-<em>cfs1</em> fusion point CTCGAGGAT CCTATG (<em>BamHI </em>cloning site in italic, native <em>cfs1</em> ATG start codon in bold) serves in transcripts as a minor RBS (ribosome binding site) to inadvertently initiate protein synthesis on the native <em>cfs1</em> start codon. The first peptide MPAHHHPSSSAPCVSFPSSSK of the native <em>Cfs1</em> protein was found in <em>E. coli </em>which is only possible if the normal <em>cfs1</em> ATG was recognized as the start codon in protein biosynthesis. The GAGG-stretch in the above DNA sequence with a 7-bases-aligned spacing to the native <em>cfs1</em> start codon features a partial Shine-Dalgarno (SD) sequence.</p>}, number={1}, journal={Current Trends in Biotechnology and Pharmacy}, author={Kües, Ursula and Kia, Sevda Haghi and Lakkireddy, Kiran and Zomorrodi, Mojtaba}, year={2021}, month={Feb.}, pages={01–14} }