title: Synthetic chimeras with orthogonal ribosomal proteins increase translation yields by recruiting mRNA for translation as measured by profiling active ribosomes.
abstract: In addition to their roles in protein biosynthesis, components of cellular ribosomes perform roles that contribute to a number of important cellular processes. Exploitation of processes has led to the use of ribosomal parts as solubility enhancer partners and purification matrices in protein expression. In this work, an engineered version of the E. coli ribosomal protein L29 (L4H2) as a fusion partner for enhancing cellular expression of proteins that are poorly expressed in bacteria was exploited. It was demonstrated that a chimeric fusion of L4H2 with various Fcγ receptors increases total expression up to 3.2-fold, relative to Fcγ receptors expressed without the fusion. Mechanistic insights using a novel application of in vivo ribosome display suggested that, although total cellular mRNA levels of L4H2-Fcγ receptor remained unchanged relative to wild-type Fcγ receptors, mRNA levels of actively translated L4H2-Fcγ transcript increased about 3.8-fold relative to actively translated levels of wild-type Fcγ transcript. Similar increases in protein expression in the context of the other proteins tested, showing the generality of this approach for proteins beyond human receptors was observed. These results extended the number of potential schemes by which orthogonal ribosomal parts can be used to enhance complex protein expression in bacterial platforms. Within a larger scope, this study features the possibility of engineering 5' tags that enhance mRNA affinity to ribosomes as strategies to augment translation. It was envisioned that the successful application of profiling active ribosomes in a highly targeted manner could be beneficial for mechanistic translation studies concerning synthesis of target proteins. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:285-293, 2016.
authors.affiliation: Inst. for Cellular & Molecular Biology, The University of Texas at Austin, Molecular Biology Building, 2500 Speedway Stop A4800, Austin, TX, 78712.
authors.forename: Seung Hee
authors.initials: SH
authors.lastname: Cho
authors.affiliation: Inst. for Cellular & Molecular Biology, The University of Texas at Austin, Molecular Biology Building, 2500 Speedway Stop A4800, Austin, TX, 78712.|McKetta Dept. of Chemical Engineering, Cockrell School of Engineering, The University of Texas at Austin, 200 E. Dean Keeton St. Stop C0400, Austin, TX, 78712.
authors.forename: Lydia M
authors.initials: LM
authors.lastname: Contreras
authors.affiliation: McKetta Dept. of Chemical Engineering, Cockrell School of Engineering, The University of Texas at Austin, 200 E. Dean Keeton St. Stop C0400, Austin, TX, 78712.
authors.forename: Sang Hyun
authors.initials: SH
authors.lastname: Ju
mesh_terms: D002678:Chimera / Q000378:metabolism*; D015202:Protein Engineering; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D012269:Ribosomal Proteins
keywords: L29; protein expression; ribosomal protein; ribosome profiling; translation enhancement