Enhanced Protein Production in Escherichia coli by Optimization of Cloning Scars at the Vector-Coding Sequence Junction
Number of Authors: 8
2015 (English)In: ACS Synthetic Biology, ISSN 0065-0897, E-ISSN 2161-5063, Vol. 4, no 9, 959-965 p.Article in journal (Refereed) Published
Protein production in Escherichia coli is a fundamental activity for a large fraction of academic, pharmaceutical, and industrial research laboratories. Maximum production is usually sought, as this reduces costs and facilitates downstream purification steps. Frustratingly, many coding sequences are poorly expressed even when they are codon-optimized and expressed from vectors with powerful genetic elements. In this study, we show that poor expression can be caused by certain nucleotide sequences (e.g., cloning scars) at the junction between the vector and the coding sequence. Since these sequences lie between the Shine-Dalgarno sequence and the start codon, they are an integral part of the translation initiation region. To identify the most optimal sequences, we devised a simple and inexpensive PCR-based step that generates sequence variants at the vector-coding sequence junction. These sequence variants modulated expression by up to WOO-fold. FACS-seq analyses indicated that low GC content and relaxed mRNA stability (AG) in this region were important, but not the only, determinants for high expression.
Place, publisher, year, edition, pages
2015. Vol. 4, no 9, 959-965 p.
protein production, translation initiation, mRNA structure, codon optimization, vector-coding sequence junction, membrane protein
IdentifiersURN: urn:nbn:se:su:diva-122500DOI: 10.1021/acssynbio.5b00033ISI: 000361858000002OAI: oai:DiVA.org:su-122500DiVA: diva2:867562