title: Re-engineering multicloning sites for function and convenience.
abstract: Multicloning sites (MCSs) in standard expression vectors are widely used and thought to be benign, non-interacting elements that exist for mere convenience. However, MCSs impose a necessary distance between promoter elements and genes of interest. As a result, the choice of cloning site defines the genetic context and may introduce significant mRNA secondary structure in the 5'-untranslated region leading to strong translation inhibition. Here, we demonstrate the first performance-based assessment of MCSs in yeast, showing that commonly used MCSs can induce dramatic reductions in protein expression, and that this inhibition is highly promoter and gene dependent. In response, we develop and apply a novel predictive model of structure-based translation inhibition to design improved MCSs for significantly higher and more consistent protein expression. In doing so, we were able to minimize the inhibitory effects of MCSs with the yeast TEF, CYC and GPD promoters. These results highlight the non-interchangeable nature of biological parts and represent the first complete, global redesign of a genetic circuit of such widespread importance as a multicloning site. The improved translational control offered by these designed MCSs is paramount to obtaining high titers of heterologous proteins in eukaryotes and to enabling precise control of genetic circuits.
authors.affiliation: Department of Chemical Engineering, The University of Texas at Austin, 1 University Station, C0400 Austin, TX 78712, USA.
authors.forename: Nathan C
authors.initials: NC
authors.lastname: Crook
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Freeman
authors.forename: Hal S
authors.initials: HS
authors.lastname: Alper
mesh_terms: D020121:5' Untranslated Regions; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon; D005822:Genetic Vectors / Q000737:chemistry*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D020121:5' Untranslated Regions; D003062:Codon