title: Expressivity tag: a novel tool for increased expression in Escherichia coli.
abstract: Protein expression in Escherichia coli is rarely trivial as low expression and insolubility are common problems. In this work we define a fusion partner, which increases expression levels similarly to the distinct function of solubility and affinity tags. This type of fusion tag we term an expressivity tag. Our work is based on earlier observations where 3' deletions of the InfB gene displays strongly increased expression levels. We have constructed progressively shortened fragments of the InfB(1-471) gene and fused gene fragments to a gfp reporter gene. A 5-fold increase in GFP expression was seen for an optimal 21 nucleotide InfB(1-21) sequence compared to gfp independently. We defined the InfB(1-21) sequence as an expressivity tag. The tag was tested for improved expression of two biotechnological important proteins streptavidin and a single chain antibody (scFv). Expression of both streptavidin and scFv(L32) was improved as evaluated by SDS-PAGE. Calculation of folding energies in the translation initiation region gave higher free energies for gfp, L32 and streptavidin when linked to InfB(1-21) than independently. InfB(1-21) did however not improve the codon usage or codon adaptation index. The expressivity tag is an important addition to the box of tools available for optimizing heterologous protein expression.
authors.affiliation: Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
authors.forename: Jon Gade
authors.initials: JG
authors.lastname: Hansted
authors.forename: Laura
authors.initials: L
authors.lastname: Pietikäinen
authors.forename: Friederike
authors.initials: F
authors.lastname: Hög
authors.forename: Hans Uffe
authors.initials: HU
authors.lastname: Sperling-Petersen
authors.forename: Kim Kusk
authors.initials: KK
authors.lastname: Mortensen
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D014176:Protein Biosynthesis; D017510:Protein Folding; D012333:RNA, Messenger / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D057127:Single-Chain Antibodies; D012995:Solubility; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018709:Statistics, Nonparametric; D013816:Thermodynamics
chemical_list: D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D049452:Green Fluorescent Proteins