title: Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression.
abstract: Autologous expression of recombinant human proteins in human cells for biomedical research and product development is often hampered by low expression yields limiting subsequent structural and functional analyses. Following RNA and codon optimization, 50 candidate genes representing five classes of human proteins--transcription factors, ribosomal and polymerase subunits, protein kinases, membrane proteins and immunomodulators--all showed reliable, and 86% even elevated expression. Analysis of three representative examples showed no detrimental effect on protein solubility while unaltered functionality was demonstrated for JNK1, JNK3 and CDC2 using optimized constructs. Molecular analysis of a sequence-optimized transgene revealed positive effects at transcriptional, translational, and mRNA stability levels. Since improved expression was consistent in HEK293T, CHO and insect cells, it was not restricted to distinct mammalian cell systems. Additionally, optimized genes represent powerful tools in functional genomics, as demonstrated by the successful rescue of an siRNA-mediated knockdown using a sequence-optimized counterpart. This is the first large-scale study addressing the influence of multiparameter optimization on autologous human protein expression.
authors.affiliation: Geneart/Life Technologies, [corrected] BioPark, Regensburg, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Fath
authors.forename: Asli Petra
authors.initials: AP
authors.lastname: Bauer
authors.forename: Michael
authors.initials: M
authors.lastname: Liss
authors.forename: Anne
authors.initials: A
authors.lastname: Spriestersbach
authors.forename: Barbara
authors.initials: B
authors.lastname: Maertens
authors.forename: Peter
authors.initials: P
authors.lastname: Hahn
authors.forename: Christine
authors.initials: C
authors.lastname: Ludwig
authors.forename: Frank
authors.initials: F
authors.lastname: Schäfer
authors.forename: Marcus
authors.initials: M
authors.lastname: Graf
authors.forename: Ralf
authors.initials: R
authors.lastname: Wagner
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003062:Codon / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D005813:Genes, Synthetic; D005821:Genetic Techniques / Q000592:standards*; D057809:HEK293 Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008322:Mammals / Q000235:genetics*; D012313:RNA / Q000235:genetics*; D034622:RNA Interference; D012015:Reference Standards; D012995:Solubility
chemical_list: D003062:Codon; D012313:RNA; D048031:JNK Mitogen-Activated Protein Kinases