title: Enhancing functional expression of heterologous proteins through random substitution of genetic codes in the 5' coding region.
abstract: Recent studies using heterologous protein expression systems suggest that synonymous codons affect not only the expression but also the properties of the expressed protein. However, practical application of this information is challenging, and to date, efforts to employ bioinformatics tools to design synonymous codon mixes have been only marginally successful. Here, we sought to enhance the functional expression of heterologous protein in Escherichia coli through completely random substitution of the first ten codons with synonymous codons, using a previously isolated exocellulase CelEdx-SF301 as the model protein. Synonymous codon variants were generated by PCR using forward primers with mixed nucleotides at the third position in each codon and a conventional reverse primer. The resulting PCR products were inserted upstream of the fluorescent protein mCherry without linkers. After transformation and cultivation, colonies exhibiting red fluorescence were selected, and the activity of SF301-mCherry fusion proteins was tested. Synonymous codon variant fusion proteins exhibited 35- to 530-fold increases in functional expression compared with wild-type controls. Unlike results from other reports, we found that the stability of mRNA secondary structure in the 5' untranslated region and codon rarity were not correlated with functional expression level. Our work demonstrates that a completely random mixed of synonymous codons effectively enhances functional expression levels without the need for amino acid substitutions.
authors.affiliation: Industrial Microbiology & Bioprocess Research Center, Integrated Biorefinery Research Institute, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181 Ipsin-gil, Jeongeup-si, Jeonbuk, 580-185, Republic of Korea.
authors.forename: Dae-Eun
authors.initials: DE
authors.lastname: Cheong
authors.forename: Kyong-Cheol
authors.initials: KC
authors.lastname: Ko
authors.forename: Yunjon
authors.initials: Y
authors.lastname: Han
authors.forename: Ho-Geun
authors.initials: HG
authors.lastname: Jeon
authors.forename: Bong Hyun
authors.initials: BH
authors.lastname: Sung
authors.forename: Geun-Joong
authors.initials: GJ
authors.lastname: Kim
authors.forename: Jong Hyun
authors.initials: JH
authors.lastname: Choi
authors.forename: Jae Jun
authors.initials: JJ
authors.lastname: Song
mesh_terms: D044602:Cellulases / Q000096:biosynthesis* / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D016296:Mutagenesis; D050505:Mutant Proteins / Q000096:biosynthesis / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D044602:Cellulases
keywords: Escherichia coli; aggregation-prone protein; functional expression; synonymous codon