Heterologous proteins expression in <i>Escherichia coli</i>: investigation of the effect of codon usage and expression host optimization

Mirzahosseini, H; Mafakheri, Samaneh; Mohammadi, Nejad; Enayati, Somayeh; Kordi, N Mortazavideh

doi:10.1142/9789814322119_0121

Cited by 3 publications

(5 citation statements)

References 18 publications

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“…Our results contradicted all previous findings which reported the N-terminal optimization of rare codons in a gene negatively affects expression levels in E. coli (35). In agreement with our results, numerous examples of increased protein expression via codon optimization are available and introduce this methodology as a common strategy for high heterologous protein expression(3637).…”

Section: Discussioncontrasting

confidence: 99%

High level expression of recombinant human growth hormone in Escherichia coli: crucial role of translation initiation region

et al. 2017

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For high-throughput production of recombinant protein in Escherichia coli (E. coli), besides important parameters such as efficient vector with strong promoter and compatible host, other important issues including codon usage, rare codons, and GC content specially at N-terminal region should be considered. In the current study, the effect of decreasing the percentage of GC nucleotides and optimizing codon usage at N-terminal region of human growth hormone (hGH) cDNA on the level of its expression in E. coli were investigated. Mutation in cDNA of hGH was performed through site-directed mutagenesis using PCR. Then, the mutant genes were amplified and cloned into the expression vector, pET-28a. The new constructs were transformed into the BL21(DE3) strain of E. coli and chemically induced for hGH expression. At the final stage, expressed proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), scanning gel densitometry, and western blot. SDS-PAGE scanning gel densitometry assay and western blot analysis revealed higher expression level of hGH by using the two new expressions constructs (mutant genes vectors with decreasing GC content and optimized-codon usage at N-terminal of cDNA) in comparison with wild gene expression vector. Obtained results demonstrated that decreasing the GC nucleotide content and optimization of codon usage at N-terminal of the hGH cDNA could significantly enhance the expression of the target protein in E. coli. Our results highlight the important role of both 5´ region of the heterologous genes in terms of codon usage and also GC content on non-host protein expression in E. coli.

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Section: Discussioncontrasting

confidence: 99%

High level expression of recombinant human growth hormone in Escherichia coli: crucial role of translation initiation region

et al. 2017

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“…In brief, amino acid sequence of native h‐PON1 (Gene bank # P27169) was used to design a gene encoding rh‐PON1 (wt) enzyme. A number of factors influence the expression of heterologous recombinant proteins, in soluble and active form, in microbial expression system . These include codon biasness, GC content and formation of a stable secondary structure by the mRNA of the target gene, and the presence of a particular “tag” in the recombinant protein .…”

Section: Resultsmentioning

confidence: 99%

“…A number of factors influence the expression of heterologous recombinant proteins, in soluble and active form, in microbial expression system. [23][24][25] These include codon biasness, GC content and formation of a stable secondary structure by the mRNA of the target gene, and the presence of a particular "tag" in the recombinant protein. [23][24][25] To express rh-PON1 enzyme in soluble and active form in Escherichia coli, a gene encoding rh-PON1 (wt) enzyme was designed using amino acid sequence of h-PON1.…”

Section: Site-directed Mutagenesis Expression and Purification Of Rhmentioning

confidence: 99%

“…[23][24][25] These include codon biasness, GC content and formation of a stable secondary structure by the mRNA of the target gene, and the presence of a particular "tag" in the recombinant protein. [23][24][25] To express rh-PON1 enzyme in soluble and active form in Escherichia coli, a gene encoding rh-PON1 (wt) enzyme was designed using amino acid sequence of h-PON1. The gene was interrogated for the presence of rare codons and mRNA secondary structure by using Visual gene developer.net and Vienna mRNA structure prediction programs.…”

Section: Site-directed Mutagenesis Expression and Purification Of Rhmentioning

confidence: 99%

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Characterization of human paraoxonase 1 variants suggest that His residues at 115 and 134 positions are not always needed for the lactonase/arylesterase activities of the enzyme

et al. 2013

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Human paraoxonase 1 (h-PON1) hydrolyzes variety of substrates and the hydrolytic activities of enzyme can be broadly grouped into three categories; arylesterase, phosphotriesterase, and lactonase. Current models of the catalytic mechanism of h-PON1 suggest that catalytic residues H115 and H134 mediate the lactonase and arylesterase activities of the enzyme. H-PON1 is a strong candidate for the development of catalytic bioscavenger for organophosphate poisoning in humans. Recently, Gupta et al. (Nat. Chem. Biol. 2011. 7, 120) identified amino acid substitutions that significantly increased the activity of chimeric-PON1 variant (4E9) against some organophosphate nerve agents. In this study we have examined the effect of these (L69G/S111T/H115W/ H134R/R192K/F222S/T332S) and other substitutions (H115W/H134R and H115W/H134R/R192K) on the hydrolytic activities of recombinant h-PON1 (rh-PON1) variants. Our results show that the substitutions resulted in a significant increase in the organophosphatase activity of all the three variants of rh-PON1 enzyme while had a variable effect on the lactonase/arylesterase activities. The results suggest that H residues at positions 115 and 134 are not always needed for the lactonase/arylesterase activities of h-PON1 and force a reconsideration of the current model(s) of the catalytic mechanism of h-PON1.

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“…The optimal codons are those that are used most frequently, and those used less frequently are termed rare or low-usage codons. If a transgene contains codon(s) rarely used by the host cell, its expression level in this host is expected to be very low [ 16 , 23 , 24 ]. If these rare codon(s) appear near the 5' end of the open reading frame, the expression of that gene will decrease even more dramatically compared to where they occur in the middle of the gene sequence [ 20 , 25 , 26 ].…”

Section: Introductionmentioning

confidence: 99%

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

Xia

et al. 2016

PLoS ONE

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Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of foreign genes in yeast and Arabidopsis.

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Heterologous proteins expression in Escherichia coli: investigation of the effect of codon usage and expression host optimization

Cited by 3 publications

References 18 publications

High level expression of recombinant human growth hormone in Escherichia coli: crucial role of translation initiation region

High level expression of recombinant human growth hormone in Escherichia coli: crucial role of translation initiation region

Characterization of human paraoxonase 1 variants suggest that His residues at 115 and 134 positions are not always needed for the lactonase/arylesterase activities of the enzyme

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

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