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.
Highlights
Heterologous expression of recombinant proteins is an indispensable process in modern biotechnology and biomedicine
To scrutinize the general validity of codon optimization for enhancing recombinant human protein expression in mammalian cell culture, we designed a large-scale study that included a broad selection of human genes
Recent advances in gene optimization combined with de novo gene synthesis allow fast and efficient construction of synthetic genes individually tailored for specific applications
Summary
Heterologous expression of recombinant proteins is an indispensable process in modern biotechnology and biomedicine. Besides the lack of posttranslational modifications or a suitable environment for membrane proteins, E. coli-mediated expression is often associated with protein misfolding or aggregation [1], imposing restrictions on large-size or oligomeric proteins. To overcome these limitations, the repertoire of expression systems for recombinant proteins was extended to gram-positive bacteria, yeast, filamentous fungi, insect cells and plants [2,3,4]. Cell lines such as CHO or HEK293 have become golden standards for high-yield production of functional recombinant human proteins
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