Selective (minimal) optimization: 10 ariginine optimized in basic region via mutagenesis.
Specifically: AGA119CGC, CGG140CGC, AGA142CGT, CGA143CGT, AGG144CGC, CGG146CGT, CGG155CGT,
CGG157CGT, AGG158CGT, AGG159CGC
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville 32610-0245, USA. email@example.com
Bacterial expression and purification of biologically active mouse c-Fos proteins by selective codon optimization
A simple strategy using selective codon optimization was devised to express mouse c-Fos protein in high levels in E. coli. Ten arginine codons located in the basic region were optimized to achieve high levels of protein expression. The c-Fos protein was purified to near homogeneity and was demonstrated to be biologically active by assaying its several biological activities.
FEBS Lett. 409(2): 269-72.
This paper presents a strategy for expressing a mouse protein (c-Fos) in E. coli. Potentially, this strategy can be applicable to other mammalian genes. c-Fos protein is part of a dimer that makes up activator protein (AP-1) and binds to the TPA response element (TRE). Unaltered mouse c-Fos DNA (CAI = 0.239) had undetectable expression in E. coli, presumably due to the presence of multiple rare Arg codons. Two approaches were used to optimize the codon usage of c-Fos for high levels of expression in E. coli. 1) Five Arg codons (at codon position 140, 142,143,144 and 146) were optimized therefore resulting in the M1 gene (CAI = 0.258); 2) then five additional Arg codons (at codon position 119,155,157,158 and 159) were optimized (M2 gene, CAI = 0.278) using oligodeoxynucleotide mutagenesis. The optimized gene was cloned into pTH6 vector and induced with IPTG for protein expression. The optimization in M1 gene did not improve the protein expression significantly. However, optimization in M2 increased protein products to comprise approximately 20% of total proteins. All analysis of the protein's properties showed that the c-Fos was biological active. Since replacing 10 Arg codons (minimal optimization) clustered around the functional domain of c-Fos was enough to dramatically increase protein expression, the authors concluded that not all of the rare codons need to be optimized to improve gene expression.