Fath, Stephan and Bauer, Asli Petra and Liss, Michael and Spriestersbach, Anne and Maertens, Barbara and Hahn, Peter and Ludwig, Christine and Schäfer, Frank and Graf, Marcus and Wagner, Ralf
Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression.
PloS one 6 (3), e17596.
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.
|Date:||3 March 2011|
|Institutions:||Medicine > Lehrstuhl für Medizinische Mikrobiologie und Hygiene|
|Gene Expression Regulation||MESH|
|Gene Knockdown Techniques||MESH|
|JNK Mitogen-Activated Protein Kinases/metabolism||MESH|
|Subjects:||600 Technology > 610 Medical sciences Medicine|
|Refereed:||Yes, this version has been refereed|
|Created at the University of Regensburg:||Yes|
|Deposited On:||11 Apr 2012 09:02|
|Last Modified:||13 Mar 2014 18:45|