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Enhancing functional expression of heterologous proteins through random substitution of genetic codes in the 5' coding region
Author(s) -
Cheong DaeEun,
Ko KyongCheol,
Han Yunjon,
Jeon HoGeun,
Sung Bong Hyun,
Kim GeunJoong,
Choi Jong Hyun,
Song Jae Jun
Publication year - 2015
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25478
Subject(s) - codon usage bias , biology , mcherry , start codon , synonymous substitution , coding region , genetics , five prime untranslated region , silent mutation , stop codon , heterologous , fusion protein , amino acid , nucleotide , gene , untranslated region , messenger rna , green fluorescent protein , mutation , recombinant dna , genome , missense mutation
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. Biotechnol. Bioeng. 2015;112: 822–826. © 2014 Wiley Periodicals, Inc.