Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study

The Structural Genomics Consortium, Old Road Campus Research Building, University of Oxford, Roosevelt Drive, Oxford OX3 7DQ, UK.
Protein Expression and Purification (Impact Factor: 1.7). 06/2008; 59(1):94-102. DOI: 10.1016/j.pep.2008.01.008
Source: PubMed


The efficiency of heterologous protein production in Escherichia coli (E. coli) can be diminished by biased codon usage. Approaches normally used to overcome this problem include targeted mutagenesis to remove rare codons or the addition of rare codon tRNAs in specific cell lines. Recently, improvements in technology have enabled cost-effective production of synthetic genes, making this a feasible alternative. To explore this option, the expression patterns in E. coli of 30 human short-chain dehydrogenase/reductase genes (SDRs) were analyzed in three independent experiments, comparing the native and synthetic (codon-optimized) versions of each gene. The constructs were prepared in a pET-derived vector that appends an N-terminal polyhistidine tag to the protein; expression was induced using IPTG and soluble proteins were isolated by Ni-NTA metal-affinity chromatography. Expression of the native and synthetic gene constructs was compared in two isogenic bacterial strains, one of which contained a plasmid (pRARE2) that carries seven tRNAs recognizing rare codons. Although we found some degree of variability between experiments, in normal E. coli synthetic genes could be expressed and purified more readily than the native version. In only one case was native gene expression better. Importantly, in most but not all cases, expression of the native genes in combination with rare codon tRNAs mimicked the behavior of the synthetic genes in the native strain. The trend is that heterologous expression of some proteins in bacteria can be improved by altering codon preference, but that this effect can be generally recapitulated by introducing rare codon tRNAs into the host cell.

Download full-text


Available from: Nicola A Burgess-Brown, Apr 28, 2015
1 Follower
68 Reads
  • Source
    • "Production of therapeutic proteins in a soluble and biologically functional form is desired for safety reasons and can significantly simplify downstream purification processes. There has been much effort spent to develop methods to improve soluble protein production from E. coli [20] [21] [22] [23] [24]. Production of a desired protein as a fusion protein in E. coli is a common approach to increase the efficiency of expression of soluble protein and to simplify its purification. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein production can be improved if methods for soluble protein expression are developed. Interferon consensus (IFN-con) is used to treat hepatitis C. IFN-con has superior activity compared to other clinically used interferon α subtypes. However IFN-con is a challenging protein to produce in a soluble form using an E.coli expression system. Here we describe the expression of soluble and active recombinant IFN-con in E. coli. The IFN-con gene sequence was optimised for expression in E.coli, which was then cloned into the Champion™ pET SUMO expression vector downstream of the SUMO fusion protein and under strong T7lac promoter. The SUMO-IFN-con fusion protein was efficiently expressed using the SHuffle™ E.coli strain and existed in soluble form as 86-88% of the total IFN-con. After removal of the SUMO fusion partner, approximately 50 mg of recombinant IFN-con of at least 98% purity (by RP-HPLC) was obtained from a 1L fermentation culture. Using an A549/EMCV antiviral assay, the specific activity of the recombinant IFN-con was determined to be 960 ×106 IU/mg as calculated to NIBSC standard for IFN-con (3x105 pfu/mL virus titre). Comparison of the antiviral activity of the produced IFN-con to IFN α -2a showed that IFN-con displays 2.8 times greater activity, which is in good agreement with what has been reported in the literature for pure protein. IFN-con expression in a soluble form from E. coli allowed us to use a simple, two-step purification process to yield highly pure and active IFN-con which is more efficient than obtaining IFN-con from inclusion bodies.
    Protein Expression and Purification 07/2014; 99. DOI:10.1016/j.pep.2014.03.009 · 1.70 Impact Factor
  • Source
    • "On the other hand, Uchijima et al. [26] supported that substitution of wild type codon usage with codons frequently found in highly expressed murine genes, generated Tc1 responses. Codon optimization can improve expression of human genes in E. coli: A multi-gene study [27]. Wang et al. [28] found a significant increase in cysC expression in E. coli produced by codon optimization techniques and they improved the protein expression from 10% to 46% based on total protein expression after codon optimization. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Purpose FimH (the adhesion fragment of type 1 fimbriae) is implicated in uropathogenic Escherichia coli (UPEC) attachment to epithelial cells through interaction with mannose. Recently, some studies have found that UPEC can thrive intracellularly causing recurrent urinary tract infection (UTI). Almost all vaccines have been designed to induce antibodies against UPEC. Yet, the humoral immune response is not potent enough to overcome neither the primary UTI nor recurrent infections. However, DNA vaccines offer the possibility of inducing cell mediated immune responses and may be a promising preventive tool. Materials and Methods In this study, we employed two different open reading frames within mammalian (mam) and wild type (wt) codons of fimH gene. Optimized fragments were cloned in pVAX-1. Expression of the protein in COS-7 was confirmed by western blot analysis after assessing pVAX/fimH(mam) and pVAX/fimH(wt). The constructs were injected to BALB/c mice at plantar surface of feet followed by electroporation. Results The mice immunized with both constructs following booster injection with recombinant FimH showed increased interferon-γ and interleukin-12 responses significantly higher than non-immunized ones (p<0.05). The immunized mice were challenged with UPEC and then the number of bacteria recovered from the immunized mice was compared with the non-immunized ones. Decreased colony count in immunized mice along with cytokine responses confirmed the promising immune response by the DNA vaccines developed in this study. Conclusion In conclusion, DNA vaccines of UPEC proteins may confer some levels of protection which can be improved by multiple constructs or boosters.
    07/2014; 3(2):185-93. DOI:10.7774/cevr.2014.3.2.185
  • Source
    • "Two strategies for solving codon usage bias have been used: codon optimization of the foreign coding sequence or increasing the availability of underrepresented tRNAs by host modification (Sorensen and Mortensen, 2005). The rationale behind codon usage optimization is to modify the rare codons in the target gene to mirror the codon usage of the host (Burgess-Brown et al., 2008; Welch et al., 2009; Menzella, 2011). The amino acid sequence of the encoded protein must not be altered in the process. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.
    Frontiers in Microbiology 04/2014; 5:172. DOI:10.3389/fmicb.2014.00172 · 3.99 Impact Factor
Show more