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.

Production of Lycopene in the Non-Carotenoid-Producing Yeast Yarrowia lipolytica

“…2909302, down to the SphI restriction site) were added to both sequences. Both constructs were further optimized by GeneOptimizer (24) and synthesized by Life Technologies GmbH (Darmstadt, Germany). The primer used in this work are summarized in Table 1.…”

High-Throughput Screening for Highly Functional RNA-Trans-Splicing Molecules: Correction of Plectin in Epidermolysis Bullosa Simplex

“…Inclusion of nuclear retention signals might have great influence on the trans-splicing efficiency, but also regarding minimization of side effects and background expression of unspliced RTMs. Codon optimization and the use of weaker promoters and enhancers can be used to increase the safety of retrovirally introduced RTMs (Baum and Schambach, 2011;Fath et al, 2011). Currently, a limiting fact is the lack of mouse models meeting the exigencies of a SMaRT approach.…”

Production of Human Norovirus Protruding Domains in <em>E. coli</em> for X-ray Crystallography

“…Moreover, remove the flexible region at the C-terminal end of the P domain (Figure 2A). Codon-optimize the DNA for E. coli expression and include BamHI (N-terminal) and NotI (C-terminal) restriction sites in order to sub-clone the P domain coding region into the pMalc2x expression vector 6,7 . Note: The P domain coding region is optimized and synthesized by a commercial service.…”