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PubMed2001

A synthetic dDAF (CD55) gene based on optimal codon usage for transgenic animals.

The human DAF (CD55) gene was chosen as a representative molecule in a xenotransplantation study. The gene was synthesized in order to adapt its codons to those which are more frequent in mammals, especially pigs, and the expression levels were then examined in Chinese hamster ovarian (CHO) cells, swine endothelial cell (SEC) and transgenic mice. A significant increase in protein production with no detectable mRNA elevation was observed in the transfectants of synthetic DAF (sDAF), compared with the wild-type DAF (wtDAF) and delta-SCR1 wild-type DAF (Delta1wtDAF). Consistent with the in vitro data, the expression of DAF in mice that carry sDAF was higher than Delta1wtDAF in many organs, especially the pancreas. The sDAF showed a high level of expression in SEC and transgenic mice, suggesting that it will be useful in the development of transgenic pigs with high levels of expression.

Animals↗
PubMed2000

Synthesis of a new Cre recombinase gene based on optimal codon usage for mammalian systems.

The origin of the Cre recombinase gene is bacteriophage P1, and thus the codon usages are different from in mammals. In order to adapt this codon usage for mammals, we synthesized a "mammalian Cre recombinase gene" and examined its expression in Chinese hamster ovarian tumor (CHO) cells. Significant increases in protein production as well as mRNA levels were observed. When the recombination efficiency was compared using CHO cell transfectants having a cDNA containing loxP sites, the "mammalian Cre recombinase gene" recombined the loxP sites much more efficiently than the wild-type Cre recombinase gene.

Amino Acid Sequence↗
PubMed2003

Optimization of codon usage is required for effective genetic immunization against Art v 1, the major allergen of mugwort pollen.

BACKGROUND: As the major allergen of mugwort pollen, Art v 1 is an important target for specific immunotherapy. However, both recombinant protein as well as a gene vaccine for Art v 1 failed to be immunogenic in mice. In order to improve immunogenicity we focused on genetic immunization because interspecific differences of codon usage have been shown as an obstacle for effective induction of immune responses with gene vaccines encoding infectious pathogens. OBJECTIVE: In order to find out, whether codon usage might also be used to improve genetic immunization with allergen genes, the response against a gene vaccine expressing the wild-type gene of Art v 1 (pCMV-wtArt) was compared with a synthetic codon-optimized vector with human codon usage (pCMV-humArt). METHODS: Balb/c mice were injected intradermally with pCMV-wtArt or pCMV-humArt. In vitro expression levels of both constructs were compared in transfection experiments. Total immunoglobulin G (IgG), IgG1, IgG2a and IgE antibodies were analyzed by enzyme-linked immunosorbent assay and the anaphylactic activity of the sera was determined by allergen-specific degranulation of rat basophil leukemia-2H3 cells. RESULTS: No immune response was detectable with the gene vaccine expressing the wildtype Art v 1, but immunization with pCMV-humArt revealed a strong and allergen-specific induction of antibody responses. The antibodies recognized both the recombinant as well as the purified natural (glycosylated) Art v 1 molecule. The response type was Th1-biased, as indicated by high levels of IgG2a antibodies. Expression analysis with B16 mouse melanoma cells transfected with pCMV-humArt or pCMV-wtArt revealed an impaired expression of the wild-type vector but normal translation after recoding. CONCLUSION: The results demonstrate that optimization of codon usage offers a simple way to improve immunogenicity and therefore should be routinely considered in the development of gene vaccines for the treatment of allergy.

Allergens↗
PubMed2006

Optimization of codon usage of poxvirus genes allows for improved transient expression in mammalian cells.

Transient expression of viral genes from certain poxviruses in uninfected mammalian cells can sometimes be unexpectedly inefficient. The reasons for poor expression levels can be due to a number of features of the gene cassette, such as cryptic splice sites, polymerase II termination sequences or motifs that lead to mRNA instability. Here we suggest that in some cases the problem of low protein expression in transfected mammalian cells may be due to inefficient codon usage. We have observed that for many poxvirus genes from the yatapoxvirus genus this deficiency can be overcome by synthesis of the gene with codon sequences optimized for expression in primate cells. This led us to examine colon usage across 2-dozen sequenced members of the Poxviridae. We conclude that codon usage is surprisingly divergent across the different Poxviridae genera but is much more conserved within a single genus. Thus, Poxviridae genera can be divided into distinct groups based on their observed codon bias. When viewed in this context, successful transient expression of transfected poxvirus genes in uninfected mammalian cells can be more accurately predicted based on codon bias. As a corollary, for specific poxvirus genes with less favorable codon usage, codon optimization can result in profoundly increased transient expression levels following transfection of uninfected mammalian cell lines.

Animals↗
PubMed1997

Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans.

The green fluorescent protein (GFP) of Aequorea victoria has been developed here as a reporter for gene expression and protein localization in Candida albicans. When wild-type (wt) GFP was expressed in C. albicans, it was not possible to detect fluorescence or a translation product for the wt protein. Since this was probably due in part to the presence of the non-canonical CTG serine codon in the Aequorea sequence, this codon was changed to the leucine codon TTG. C. albicans cells expressing this construct contained GFP mRNA but were non-fluorescent and contained no detectable translation product. Hence a codon-optimized GFP gene was constructed in which all of the 239 amino acids are encoded by optimal codons for C. albicans. In this gene were also incorporated two previously identified mutations in the chromophore that increase GFP fluorescence. C. albicans cells expressing this yeast-enhanced GFP gene (yEGFP3) are fluorescent and contain GFP protein. yEGFP3 can be used as a versatile reporter of gene expression in C. albicans and Saccharomyces cerevisiae and the optimized GFP described here should have broad applications in these and other fungal species.

Animals↗
PubMed2005

Production of (10E,12Z)-conjugated linoleic acid in yeast and tobacco seeds.

The polyenoic fatty acid isomerase from Propioniumbacterium acnes (PAI) was expressed in E. coli and biochemically characterized. PAI catalyzes the isomerization of a methylene-interrupted double bond system to a conjugated double bond system, creating (10E,12Z)-conjugated linoleic acid (CLA). PAI accepted a wide range of free polyunsaturated fatty acids as substrates ranging from 18:2 fatty acids to 22:6, converting them to fatty acids with two or three conjugated double bonds. For expression of PAI in yeast the PAI-sequence encoding 20 N-terminal amino acid residues was altered for optimal codon usage, yielding codon optimized PAI (coPAI). The percentage of 10,12-CLA of total esterified fatty acids was 8 times higher in yeast transformed with coPAI than in cells transformed with PAI. CLA was detected in amounts up to 5.7% of total free fatty acids in yeast transformed with coPAI but none was detected in yeast transformed with PAI. PAI or coPAI under the control of the constitutive CaMV 35S promoter or the seed-specific USP promoter was transformed into tobacco plants. CLA was only detected in seeds in coPAI-transgenic plants. The amount of CLA detected in esterified fatty acids was up to 0.3%, in free fatty acids up to 15%.

Actinomycetales↗
PubMed1982

Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes.

By considering the nucleotide sequence of several highly expressed coding regions in bacteriophage MS2 and mRNAs from Escherichia coli, it is possible to deduce some rules which govern the selection of the most appropriate synonymous codons NNU or NNC read by tRNAs having GNN, QNN or INN as anticodon. The rules fit with the general hypothesis that an efficient in-phase translation is facilitated by proper choice of degenerate codewords promoting a codon-anticodon interaction with intermediate strength (optimal energy) over those with very strong or very weak interaction energy. Moreover, codons corresponding to minor tRNAs are clearly avoided in these efficiently expressed genes. These correlations are clearcut in the normal reading frame but not in the corresponding frameshift sequences +1 and +2. We hypothesize that both the optimization of codon-anticodon interaction energy and the adaptation of the population to codon frequency or vice versa in highly expressed mRNAs of E. coli are part of a strategy that optimizes the efficiency of translation. Conversely, codon usage in weakly expressed genes such as repressor genes follows exactly the opposite rules. It may be concluded that, in addition to the need for coding an amino acid sequence, the energetic consideration for codon-anticodon pairing, as well as the adaptation of codons to the tRNA population, may have been important evolutionary constraints on the selection of the optimal nucleotide sequence.

Anticodon↗
PubMed2006

The evolution of biased codon and amino acid usage in nematode genomes.

Despite the degeneracy of the genetic code, whereby different codons encode the same amino acid, alternative codons and amino acids are utilized nonrandomly within and between genomes. Such biases in codon and amino acid usage have been demonstrated extensively in prokaryote genomes and likely reflect a balance between the action of mutation, selection, and genetic drift. Here, we quantify the effects of selection and mutation drift as causes of codon and amino acid-usage bias in a large collection of nematode partial genomes from 37 species spanning approximately 700 Myr of evolution, as inferred from expressed sequence tag (EST) measures of gene expression and from base composition variation. Average G + C content at silent sites among these taxa ranges from 10% to 63%, and EST counts range more than 100-fold, underlying marked differences between the identities of major codons and optimal codons for a given species as well as influencing patterns of amino acid abundance among taxa. Few species in our sample demonstrate a dominant role of selection in shaping intragenomic codon-usage biases, and these are principally free living rather than parasitic nematodes. This suggests that deviations in effective population size among species, with small effective sizes among parasites, are partly responsible for species differences in the extent to which selection shapes patterns of codon usage. Nevertheless, a consensus set of optimal codons emerges that is common to most taxa, indicating that, with some notable exceptions, selection for translational efficiency and accuracy favors similar sets of codons regardless of the major codon-usage trends defined by base compositional properties of individual nematode genomes.

Amino Acids↗
PubMed2001

Why are translationally sub-optimal synonymous codons used in Escherichia coli?

Natural selection favors certain synonymous codons which aid translation in Escherichia coli, yet codons not favored by translational selection persist. We use the frequency distributions of synonymous polymorphisms to test three hypotheses for the existence of translationally sub-optimal codons: (1) selection is a relatively weak force, so there is a balance between mutation, selection, and drift; (2) at some sites there is no selection on codon usage, so some synonymous sites are unaffected by translational selection; and (3) translationally sub-optimal codons are favored by alternative selection pressures at certain synonymous sites. We find that when all the data is considered, model 1 is supported and both models 2 and 3 are rejected as sole explanations for the existence of translationally sub-optimal codons. However, we find evidence in favor of both models 2 and 3 when the data is partitioned between groups of amino acids and between regions of the genes. Thus, all three mechanisms appear to contribute to the existence of translationally sub-optimal codons in E. coli.

Codon↗
PubMed1998

Optimization of codon usage of plasmid DNA vaccine is required for the effective MHC class I-restricted T cell responses against an intracellular bacterium.

In an attempt to study codon usage effects of DNA vaccines on the induction of MHC class I-restricted T cell responses against an intracellular bacterium, Listeria monocytogenes, we designed two plasmid DNA vaccines encoding an H-2Kd-restricted epitope of listeriolysin O (LLO) of L. monocytogenes, LLO 91-99. One DNA vaccine, p91wt, carries the wild-type DNA sequence encoding LLO 91-99, and the other one, p91mam, possesses the altered DNA sequence in which the codon usage was optimized for murine system. Our read-through analyses with LLO 91-99/luciferase fusion genes confirmed that the optimized 91mam DNA sequence showed extremely higher translation efficiency than the wild-type sequence in murine cells. Consistent with this, i.m. injections of p91mam, but not of p91wt, into BALB/c mice were capable of inducing specific CTL- and IFN-gamma-producing CD8+ T cells able to confer partial protection against listerial challenge. Taken together, these observations suggest that optimization of codon should be taken into consideration in the construction of DNA vaccines against nonviral pathogens.

Animals↗
PubMed2005

Comparative studies on codon usage pattern of chloroplasts and their host nuclear genes in four plant species.

A detailed comparison was made of codon usage of chloroplast genes with their host (nuclear) genes in the four angiosperm species Oryza sativa, Zea mays, Triticum aestivum and Arabidopsis thaliana. The average GC content of the entire genes, and at the three codon positions individually, was higher in nuclear than in chloroplast genes, suggesting different genomic organization and mutation pressures in nuclear and chloroplast genes. The results of Nc-plots and neutrality plots suggested that nucleotide compositional constraint had a large contribution to codon usage bias of nuclear genes in O. sativa, Z. mays, and T. aestivum, whereas natural selection was likely to be playing a large role in codon usage bias in chloroplast genomes. Correspondence analysis and chi-test showed that regardless of the genomic environment (species) of the host, the codon usage pattern of chloroplast genes differed from nuclear genes of their host species by their AU-richness. All the chloroplast genomes have predominantly A- and/or U-ending codons, whereas nuclear genomes have G-, C- or U-ending codons as their optimal codons. These findings suggest that the chloroplast genome might display particular characteristics of codon usage that are different from its host nuclear genome. However, one feature common to both chloroplast and nuclear genomes in this study was that pyrimidines were found more frequently than purines at the synonymous codon position of optimal codons.

Arabidopsis↗
PubMed2005

The Synthetic Gene Designer: a flexible web platform to explore sequence manipulation for heterologous expression.

"Codon optimization" is a general approach to improving heterologous expression where genes are moved from their native genomes into alternatives that exhibit different patterns of codon usage. However, despite reports of successful manipulations and the existence of stand-alone codon optimization software packages or commercial services that offer to redesign genes, the scientific community lacks any systematic understanding of what exactly it means to optimize codon usage. Thus we present a bona fide web application, the "Synthetic Gene Designer," which contrasts with existing software by providing a centralized, free, and transparent platform for the broader scientific community to develop knowledge about synthetic gene design. Consistent with this goal, our software is associated with a moderated e-forum that promotes discussion of synthetic gene design and offers technical support. In addition, the Synthetic Gene Designer presents enhanced functionality over existing software options: for example, it enables users to work with non-standard genetic codes, with user-defined patterns of codon usage and an expanded range of methods for codon optimization. The Synthetic Gene Designer, together with on-line tutorials and the forum, is available at .

Animals↗
PubMed2006

[Codon usage of Chinese hamster ovary cells.].

BACKGROUND: To investigate the optimal codons of dihydrofolate reductase (dhfr) minus Chinese hamster ovary cells (CHO dhfr-). METHODS: A cDNA library of CHO dhfr- containing high abundence mRNA was constructed and protein-coding sequences were obtained after identification and analysis. Codon frequence of CHO dhf- was compared with that of Chinese hamster in CUTG database. Then codon usage variation among cDNA was investigated using correspondence analysis (COA). RESULTS: Fifty qualified cDNAs from CHO dhfr- were selected, which encodes proteins of high abundence. Comparing with the codon frequence of Chinese hamster, the highest frequence of synonymous codons for amino acids in CHO dhfr- cells were the same as Chinese hamster except that of Arg and Pro. This method of COA identifies the first main factor which can account for the largest fractions (14.7%) of variation among cDNAs. Twenty-two synonymous codons were identified as the optimal codons of CHO cell. CONCLUSION: CHO dhfr- cell has its own optimal codons, it is suggested that codon bias is one of reasons for functional diversity of different mammal cells and it is an effective stratagy to modification of the codon usage of the foreign gene according to the optimal codons of CHO dhfr- to increase the production of foreign gene.

Animals↗
PubMed2004

Codon bias as a factor in regulating expression via translation rate in the human genome.

We study the interrelations between tRNA gene copy numbers, gene expression levels and measures of codon bias in the human genome. First, we show that isoaccepting tRNA gene copy numbers correlate positively with expression-weighted frequencies of amino acids and codons. Using expression data of more than 14,000 human genes, we show a weak positive correlation between gene expression level and frequency of optimal codons (codons with highest tRNA gene copy number). Interestingly, contrary to non-mammalian eukaryotes, codon bias tends to be high in both highly expressed genes and lowly expressed genes. We suggest that selection may act on codon bias, not only to increase elongation rate by favoring optimal codons in highly expressed genes, but also to reduce elongation rate by favoring non-optimal codons in lowly expressed genes. We also show that the frequency of optimal codons is in positive correlation with estimates of protein biosynthetic cost, and suggest another possible action of selection on codon bias: preference of optimal codons as production cost rises, to reduce the rate of amino acid misincorporation. In the analyses of this work, we introduce a new measure of frequency of optimal codons (FOP'), which is unaffected by amino acid composition and is corrected for background nucleotide content; we also introduce a new method for computing expected codon frequencies, based on the dinucleotide composition of the introns and the non-coding regions surrounding a gene.

Algorithms↗
PubMed2003

Codon usage optimization of HIV type 1 subtype C gag, pol, env, and nef genes: in vitro expression and immune responses in DNA-vaccinated mice.

Codon usage optimization of human immunodeficiency virus type 1 (HIV-1) structural genes has been shown to increase protein expression in vitro as well as in the context of DNA vaccines in vivo; however, all optimized genes reported thus far are derived from HIV-1 (group M) subtype B viruses. Here, we report the generation and biological characterization of codon usage-optimized gag, pol, env (gp160, gp140, gp120), and nef genes from a primary (nonrecombinant) HIV-1 subtype C isolate. After transfection into 293T cells, optimized subtype C genes expressed one to two orders of magnitude more protein (as determined by immunoblot densitometry) than the corresponding wild-type constructs. This effect was most pronounced for gp160, gp140, Gag, and Pol (>250-fold), but was also observed for gp120 and Nef (45- and 20-fold, respectively). Optimized gp160- and gp140-derived glycoproteins were processed, incorporated into virus particles, and mediated virus entry when expressed in trans to complement an env-minus HIV-1 provirus. Mice immunized with optimized gp140 DNA developed antibody as well as CD4+ and CD8+ T cell immune responses that were orders of magnitude greater than those of mice immunized with wild-type gp140 DNA. These data confirm and extend previous studies of codon usage optimization of HIV-1 genes to the most prevalent group M subtype. Our panel of matched optimized and wild-type subtype C genes should prove valuable for studies of protein expression and function, the generation of subtype-specific immunological reagents, and the production of DNA-based sub-unit vaccines directed against a broader spectrum of viruses.

AIDS Vaccines↗
PubMed2005

Redesign, synthesis and functional expression of the 6-deoxyerythronolide B polyketide synthase gene cluster.

A generic design of Type I polyketide synthase genes has been reported in which modules, and domains within modules, are flanked by sets of unique restriction sites that are repeated in every module [1]. Using the universal design, we synthesized the six-module DEBS gene cluster optimized for codon usage in E. coli, and cloned the three open reading frames into three compatible expression vectors. With one correctable exception, the amino acid substitutions required for restriction site placements were compatible with polyketide production. When expressed in E. coli the codon-optimized synthetic gene cluster produced significantly more protein than did the wild-type sequence. Indeed, for optimal polyketide production, PKS expression had to be down-regulated by promoter attenuation to achieve balance with expression of the accessory proteins needed to support polyketide biosynthesis.

Escherichia coli↗
PubMed2006

High-level expression of human beta-defensin-2 gene with rare codons in E. coli cell-free system.

Human beta-defensin-2 (hBD2) is A small cationic peptide with A broad range of antimicrobial activity. An E. coli cell-free system was employed to express the hBD2 fusion protein by using the hBD2 gene with 14 rare codons. The results showed that the expression level of trxA-hBD2 fusion protein was 0.35 mg/ml, which is the same as that obtained with A synthetic codon-optimized gene. By using another fusion partner (GFP), similar high-level expression was also achieved in this cell-free system. This meant that human beta-defensin-2 gene could be directly used to express hBD2 fusion protein efficiently in an E. coli cell-free system without the optimization of codons. The expression level of hBD2 fused with thioredoxin could be further improved up to 2.0 mg/ml by adopting A continuous exchange cell-free system. A simple one-stage affinity purification procedure was also developed to recover this fusion protein efficiently.

Cell-Free System↗
PubMed2006

[Expression and secretion of human bone morphogenetic protein-7 in Pichia pastoris].

The synonymous codons are used in a highly non-random manner in hosts of widely divergent species, which is termed "codon usage bias". Several reports suggest that codon usage bias sometimes frustrate attempts to express high levels of exogenous genes. In this study, we attempted to express mature peptide of human bone morphogenetic protein-7(hBMP7), with optimized codons in P. pastoris expression system. Three low-usage ARG codons (CGG or CGA) of gene fragment coding the mature peptide of hBMP7 have been successfully converted into P. pastoris-preferred ARG codons (AGA) by overlap extension PCR-based multiple-site-directed mutagenesis for a high level expression of hBMP7 mature peptide. The present results showed that the production level (25.45 mg/L) of codon-optimized hbmp7 had a remarkably improvement of 4.6-fold relative to that (5.5 mg/L) of non-codon-optimized hbmp7. Furthermore, a strain haboring multi-copy of codon-optimized hbmp7 expression cassette was screened, and showed a increased level of expression with 2-fold more potent than the single-copy one. The recombinant hBMP7 mature peptide were produced as a 18 kD monomer proteins, and were easily purified from culture supernatants by using ion-exchange chromatography. Functional assay demonstrated that rhBMP7 could induce ectopic cartilage formation, although its inductive ability was much less active than CHO cell-derived hBMP7.

Animals↗