title: Enhanced Protein Production in Escherichia coli by Optimization of Cloning Scars at the Vector-Coding Sequence Junction.
abstract: 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 1000-fold. FACS-seq analyses indicated that low GC content and relaxed mRNA stability (ΔG) in this region were important, but not the only, determinants for high expression.
authors.forename: Kiavash
authors.initials: K
authors.lastname: Mirzadeh
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , DK-2970 Hørsholm, Denmark.
authors.forename: Virginia
authors.initials: V
authors.lastname: Martínez
authors.forename: Stephen
authors.initials: S
authors.lastname: Toddo
authors.forename: Suchithra
authors.initials: S
authors.lastname: Guntur
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , DK-2970 Hørsholm, Denmark.
authors.forename: Markus J
authors.initials: MJ
authors.lastname: Herrgård
authors.forename: Arne
authors.initials: A
authors.lastname: Elofsson
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , DK-2970 Hørsholm, Denmark.
authors.forename: Morten H H
authors.initials: MH
authors.lastname: Nørholm
authors.forename: Daniel O
authors.initials: DO
authors.lastname: Daley
mesh_terms: D003001:Cloning, Molecular; D018387:Codon, Initiator / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D059014:High-Throughput Nucleotide Sequencing; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D020871:RNA Stability; D017422:Sequence Analysis, DNA
chemical_list: D018387:Codon, Initiator; D004269:DNA, Bacterial
keywords: codon optimization; mRNA structure; membrane protein; protein production; translation initiation; vector−coding sequence junction