Enhancing functional expression of codon‐optimized heterologous enzymes in Escherichia coli BL21(DE3) by selective introduction of synonymous rare codons

Abstract

Rare codon in a heterologous gene may cause premature termination of protein synthesis, misincorporation of amino acids, and/or slow translation of mRNA, decreasing the heterologous protein expression. However, its hypothetical function pertaining to functional protein folding has been barely reported. Here, we investigated the effects of selective introduction of synonymous rare codons (SRCs) to two codon-optimized (i.e., rare codon-free) genes sucrose phosphorylase (SP) gene from Thermoanaerobacterium thermosaccharolyticum and amidohydrolase gene from Streptomyces caatingaensis on their expression levels in Escherichia coli BL21(DE3). We investigated the introduction of a single SRC to the coding regions of alpha-helix, beta-strand, or linker in the first half of rare codon-free sp and ah gene. The introduction of a single SRC in the beginning of the coding regions of beta-strand greatly enhanced their soluble expression levels as compared to the other regions. Also, we applied directed evolution to test multi-SRC-containing sp gene mutants for enhanced soluble SP expression levels. To easily identify the soluble SP expression level of colonies growing on Petri dishes, mCherry fluorescent protein was used as a SP-folding reporter when it was fused to the 3' end of the sp gene mutant libraries. After three rounds of screening, the best sp gene mutant containing nine SRCs exhibited an approximately six-fold enhancement in soluble protein expression level as compared to the wild-type and rare codon-free sp control. This study suggests that the selective introduction of SRCs can attenuate translation at specific points and such discontinuous attenuation can temporally separate the translation of segments of the peptide chains and actively coordinates their co-translational folding, resulting in enhanced functional protein expression. Biotechnol. Bioeng. 2017;114: 1054-1064. © 2016 Wiley Periodicals, Inc.

Advances in COVID-19 mRNA vaccine development

“…To improve the safety, efficacy, and stability of mRNA vaccines, researchers usually performed codon optimization 172 , 239 – 243 on the antigen-coding sequence to enhance translation efficiency. Optimization of mRNA secondary structure 244 , 245 and stability can be achieved by increasing the GC content of the coding sequence. 246 – 248 Codon preference varies extensively in different organisms, 249 therefore adjusting the balance between codon usage frequency and host tRNA availability can significantly improve translation efficiency and in vivo expression of the target antigen.…”

mRNA vaccine: a potential therapeutic strategy

“…As mentioned above, replacing the mRNA sequence with rare codons and introducing modified nucleotides are likely to change the mRNA structure, the translational accuracy [60] and the protein-folding mechinery [61]. These changes further influence the type, intensity and specificity of immune responses.…”

Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency

“…The 5′-UTR/3′-UTR, 5′ cap, the poly(A) tail, a rare codon and G:C content can be optimized to improve mRNA stability. The mRNA structure, translational efficiency, and protein-folding machinery are most likely to be altered by substituting the mRNA sequence with rare codons and inserting modified nucleotides (Zhong et al 2017 ). The intensity and specificity of the immune responses are expected to be influenced by these changes.…”