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Results for “codon optimization”

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At least 19 recordsLinked to original sources

PubMed2003

Codon optimizer: a freeware tool for codon optimization.

Selection plays a major role in the determination of codon usage in all organisms studied so far. In highly expressed genes, a narrow set of codons is used and these codons correspond to the more abundant tRNA species. This minimizes the risk of tRNA depletion during translation. In fact, the codons in a gene may be true bottlenecks, especially in cases where foreign genes are expressed in a host in which the usage of codons in highly expressed genes does not resemble the usage of codons in the species from which the foreign gene originates. In such cases, it has been shown that substitution of rare codons in the introduced gene may increase the yield dramatically. In addition, replacement of rare codons might decrease the chance of misincorporation and protect the protein from premature turnover. Here, a piece of software is announced that calculates a codon-optimized sequence of any gene based on knowledge of highly expressed genes of a host. In addition, it calculates the codon adaptation index of the gene and identifies internal type II restriction sites of the optimized sequence. The program runs under Windows and is available as freeware for use in academia.

Animals↗

PubMed2004

Codon optimization, expression, and characterization of recombinant lumbrokinase in goat milk.

Lumbrokinase is an important fibrinolytic enzyme derived from earthworm. Although its cDNA has been isolated and sequenced, there is still no report on expression of the lumbrokinase due to unknown reasons. To determine the elements affecting the expression of lumbrokinase, two copies of a lumbrokinase cDNA(w) obtained by RT-PCR and a synthesized lumbrokinase cDNA(m) with optimized codons were cloned into a mammary-gland-specific expression vector pIbCP. The pIbCP-LK-LK vector preparations were directly injected in the lactating goat mammary glands. Results showed that both LK-w and LK-m were successfully expressed in goat milk. The fibrinolytic activity of the LK-w in milk was 225,000 +/- 13,200 tPA units/L, while that of the LK-m was 550,000 +/- 21,600 tPA units/L, indicating that the codon optimization plays an important role in improving the lumbrokinase expression. The molecular weight of the recombinant lumbrokinase is 31.8 kDa. The main physiochemical features of the recombinant lumbrokinase, including temperature stability, pH resistance, and sensitivity to pepsin, were also clarified. This is the first report on expression and characterization of a genetically engineered lumbrokinase.

Animals↗

PubMed2007

Codon optimized Thermobifida fusca hydrolase secreted by Bacillus megaterium.

Production and secretion of a 28,172 Da hydrolase from Thermobifida fusca (TFH) in Bacillus megaterium MS941 and WH323 was investigated in shake flask and pH controlled bioreactors. Successful production of heterologous TFH was achieved by adapting the original tfh gene to the optimal codon usage of B. megaterium. A codon adaption index close to one was reached. The codon optimized tfh was cloned into an open reading frame with DNA sequence for the N-terminal signal peptide of B. megaterium lipase A and a C-terminal His(6)-tag, all under the control of a xylose inducible promoter. Successful TFH production and secretion were observed using batch reactor cultivations with complex medium. Expression of the tfh gene from the P(xylA) promoter and secretion of produced TFH were compared in detail to batch reactor cultivations with semi-defined growth medium. For the first time, significant TFH secretion was achieved using a semi-defined medium in glucose limited fed batch cultivations yielding 10-fold higher cell densities compared to LB medium cultivation. Comparable volumetric TFH activities were obtained for both cultivation strategies. Surprisingly, measured specific TFH activities exhibited drastic discrepancies between preparations from LB and semi-defined medium grown B. megaterium. TFH recovery by Ni-chelate affinity chromatography resulted in higher purification factors when LB medium was used. These results indicated that secreted TFH is favorably produced by batch cultures of B. megaterium WH323 in LB medium.

Actinomycetales↗

PubMed2003

HIV-1 gag expression is quantitatively dependent on the ratio of native and optimized codons.

There is a significant variation of codon usage bias among different species and even among genes within the same organisms. Codon optimization, this is, gene redesigning with the use of codons preferred for the specific expression system, results in improved expression of heterologous genes in bacteria, plants, yeast, mammalian cells, and transgenic animals. The mechanisms preventing expression of genes with rare or low-usage codons at adequate levels are not completely elucidated. Human immunodeficiency virus (HIV) represents an interesting model for studying how differences in codon usage affect gene expression in heterologous systems. Construction of synthetic genes with optimized codons demonstrated that the codon-usage effects might be a major impediment to the efficient expression of HIV gag/pol and env gene products in mammalian cells. According to another hypothesis, the poor expression of HIV structural proteins even without HIV context is attributed to the so-called cis-acting inhibitory elements (INS), which are located within the protein-coding region. They consist of AU-rich sequences and may be inactivated through the introduction of multiple mutations over the large regions of gag gene. In our work, we evaluated expression of hybrid HIV-1 gag mRNAs where wild-type (A-rich) gag sequences were combined with artificial sequences. In such "humanized" gag fragments with adapted codon usage, AT-content was significantly reduced in favor of G and C nucleotides without any changes in protein sequence. We show that wild-type gag sequences negatively influence expression of gag-reporter, and the addition of fragments with optimized codons to gag mRNA partially rescues its expression. The results demonstrate that the expression of HIV-1 gag is determined by the ratio of optimized and rare codons within mRNA. Our data also indicates that some wtgag fragments counteract the influence of the other wtgag sequences, which cause the inhibition of gag expression. The presented data do not contradict the concept of INS; yet, it makes the definition of INS more complex. This supports the idea of a broader role of the selected codon usage in influencing the expression of HIV proteins in mammalian cells.

Codon↗

PubMed2005

Mycobacterial codon optimization enhances antigen expression and virus-specific immune responses in recombinant Mycobacterium bovis bacille Calmette-Guérin expressing human immunodeficiency virus type 1 Gag.

Although its potential for vaccine development is already known, the introduction of recombinant human immunodeficiency virus (HIV) genes to Mycobacterium bovis bacille Calmette-Guérin (BCG) has thus far elicited only limited responses. In order to improve the expression levels, we optimized the codon usage of the HIV type 1 (HIV-1) p24 antigen gene of gag (p24 gag) and established a codon-optimized recombinant BCG (rBCG)-p24 Gag which expressed a 40-fold-higher level of p24 Gag than did that of nonoptimized rBCG-p24 Gag. Inoculation of mice with the codon-optimized rBCG-p24 Gag elicited effective immunity, as evidenced by virus-specific lymphocyte proliferation, gamma interferon ELISPOT cell induction, and antibody production. In contrast, inoculation of animals with the nonoptimized rBCG-p24 Gag induced only low levels of immune responses. Furthermore, a dose as small as 0.01 mg of the codon-optimized rBCG per animal proved capable of eliciting immune responses, suggesting that even low doses of a codon-optimized rBCG-based vaccine could effectively elicit HIV-1-specific immune responses.

AIDS Vaccines↗

PubMedDate not supplied

UpGene: Application of a web-based DNA codon optimization algorithm.

Although DNA codon optimization is a standard molecular biology strategy to overcome poor gene expression, to date no public software exists to facilitate this process. Among the uses of codon optimization, human immunodeficiency virus (HIV) vaccine development represents one of the most difficult challenges. A key obstacle to an effective DNA-based vaccine is the low-level expression of HIV genes in mammalian cells, which is due primarily to the instability of HIV mRNAs resulting from AU-rich elements and rare codon usage. In this report we describe the development of a DNA optimization algorithm integrated with a PCR primer design program to redesign specific coding sequences for maximal gene expression. Using this algorithm combination, together with PCR-based gene assembly, we have successfully optimized gene sequences for simian immunodeficiency virus (SIV) strain mac239 structural antigenic proteins gag and env, resulting in high-level gene expression in eukaryotic cells. Our findings demonstrate that our user-friendly algorithm is a valuable tool for DNA-based HIV vaccine development. Moreover, it can be used to optimize any other genes of interest and is freely available online at http://www.vectorcore.pitt.edu/upgene.html.

Algorithms↗

PubMed2005

Codon optimization of bacterial luciferase (lux) for expression in mammalian cells.

Expression of the bacterial luciferase (lux) system in mammalian cells would culminate in a new generation of bioreporters for in vivo monitoring and diagnostics technology. Past efforts to express bacterial luciferase in mammalian cells have resulted in only modest gains due in part to low overall expression of the bacterial genes. To optimize expression, we have designed and synthesized codon-optimized versions of the luxA and luxB genes from Photorhabdus luminsecens. To evaluate these genes in vivo, stable HEK293 cell lines were created harboring wild type luxA and luxB (WTA/WTB), codon-optimized luxA and wild type luxB (COA/WTB), and codon-optimized versions of both luxA and luxB genes (COA/COB). Although mRNA levels within these clones remained approximately equal, LuxA protein levels increased significantly after codon optimization. On average, bioluminescence levels were increased by more than six-fold [5x10(5) vs 2.9x10(6) relative light units (RLU)/mg total protein] with the codon-optimized luxA and wild type luxB. Bioluminescence was further enhanced upon expression of both optimized genes (2.7x10(7) RLU/mg total protein). These results show promise toward the potential development of an autonomous light generating lux reporter system in mammalian cells.

Animals↗

PubMed2002

[Optimized codon usage enhances the expression and immunogenicity of DNA vaccine encoding the HPV 6b E7 gene].

OBJECTIVE: To analyze the influence of optimal codon usage on the expression levels and immunogenicity of DNA vaccines, encoding the human papillomavirus type 6b (HPV 6b) E7 gene. METHODS: The full length E7 gene of HPV 6b was modified to substitute human preferred codon for rarely used codon, and three mutations were introduced into the pRB binding site of HPV 6b E7 to eliminate its transformation potential. The codon optimized and mutated E7 gene (hu-mE7) were cloned into the Kpn I and EcoR I site of the pcDNA3 mammalian expression vector, the in vitro expression of the hu-mE7 gene and the immunogenicity of hu-mE7 DNA vaccine were compared with the wt-E7gene. RESULTS: The in vitro expression of pcDNA3-hu-mE7 was much higher than the classical wt-E7 plasmid in monkey COS-1 cell line. Mice immunized intramuscularly with the pcDNA3-hu-mE7 showed that the codon modified E7 gene induced a stronger IFN-gamma ratios than the wt-E7 gene. CONCLUSIONS: These results suggest that the optimized codon usage contributes to the enhancement of gene expression and immunogenicity of HPV 6b E7 gene.

Animals↗

PubMed1989

Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do 'prefer' optimal codons.

Codon usage patterns in the slime mould Dictyostelium discoideum have been re-examined (a total of 58 genes have been analysed). Considering the extreme A + T-richness of this genome (G + C = 22%), there is a surprising degree of codon usage variation among genes. For example, G + C content at silent sites varies from less than 10% to greater than 30%. It was previously suggested [Warrick, H.M. and Spudich, J.A. (1988) Nucleic Acids Res. 16: 6617-6635] that highly expressed genes contain fewer 'optimal' codons than genes expressed at lower levels. However, it appears that the optimal codons were misidentified. Multivariate statistical analysis shows that the greatest variation among genes is in relative usage of a particular subset of codons (about one per amino acid), many of which are C-ending. We have identified these as optimal codons, since (i) their frequency is positively correlated with gene expression level, and (ii) there is a strong mutation bias in this genome towards A and T nucleotides. Thus, codon usage in D. discoideum can be explained by a balance between the forces of mutational bias and translational selection.

Codon↗

PubMed2003

Effects of codon-optimization on protein expression by the human herpesvirus 6 and 7 U51 open reading frame.

Codon-optimization refers to the alteration of gene sequences, to make codon usage match the available tRNA pool within the cell/species of interest. Codon-optimization has emerged as a powerful tool to increase protein expression by genes from small RNA and DNA viruses, which commonly contain overlapping reading frames as well as structural elements that are embedded within coding regions; these features are not widespread among large DNA viruses. We therefore examined whether codon-optimization might influence protein expression from a herpesvirus gene. We focused on the U51 gene from human herpesviruses-6 and -7, which was cloned in both native and codon-optimized form, with an N-terminal HA epitope tag to allow protein detection. Codon-optimization was associated with a profound (10-100 fold) increase in U51 expression in human (293A, HSG, K562) or hamster (CHO) cell lines, suggesting this may represent a valuable tool to facilitate functional studies on recalcitrant herpesvirus genes. Finally, it is postulated that the suboptimal expression of native U51 may reflect a regulatory mechanism that controls viral gene expression.

Animals↗

PubMed2004

Codon optimization reveals critical factors for high level expression of two rare codon genes in Escherichia coli: RNA stability and secondary structure but not tRNA abundance.

Expression patterns in Escherichia coli of two small archaeal proteins with a natural content of about 30% rare codons were analyzed. The proteins, a histone-like protein from Sulfolobus shibatae (Ssh10), and a glutaredoxin-like protein from Methanobacterium thermoautotrophicum (mtGrx), were produced with expression plasmids encoding wild-type genes, codon-optimized synthetic, and GST-fusion genes. These constructs were expressed in BL21 (DE3), its LysS derivative, and modified strains carrying copies for rare codon tRNAs or deletions in the RNAseE gene. Both Ssh10 and mtGrx expression levels were constitutively high in BL21(DE3) and its derivatives, with the exception of the LysS phenotype, which prevented high level expression of the Ssh10 wild-type gene. Surprisingly, a codon-optimized mtGrx gene construct displayed undetectable levels of protein production. The translational block observed with the synthetic mtGrx gene could be circumvented by using a synthetic mtGrx-glutathione S-transferase (GST) fusion construct or by in vitro translation. Taken together, the results underscore the importance of mRNA levels and RNA stability, but not necessarily tRNA abundance for efficient heterologous protein production in E. coli.

Archaeal Proteins↗

PubMed2006

Hemagglutinin (HA) proteins from H1 and H3 serotypes of influenza A viruses require different antigen designs for the induction of optimal protective antibody responses as studied by codon-optimized HA DNA vaccines.

Effective antibody responses provide crucial immunity against influenza virus infection. The hemagglutinin (HA) protein is the major target of protective antibody responses induced by viral infection and by vaccination with both inactivated and live-attenuated flu vaccines, but knowledge about the optimal designs of protective HA antigens from different flu serotypes is still limited. In this study, we have significantly improved the immunogenicity of HA-expressing DNA vaccines by using codon-optimized HA sequences for either an H1 serotype (A/NewCal/20/99) or an H3 serotype (A/Panama/2007/99) human influenza A virus and then used these constructs as model antigens to identify the optimal HA antigen designs to elicit high-level protective antibody responses. Two forms of HA antigen, a wild-type, full-length HA and a secreted form with transmembrane (TM) domain-truncated HA, were produced. Both forms of HA DNA vaccines, from either H1 or H3 serotypes, were able to elicit high levels of HA-specific immunoglobulin G responses in immunized rabbits as measured by enzyme-linked immunosorbent assay. Interestingly, the abilities of H1 HA and H3 HA antigens to elicit hemagglutination inhibition (HI) and neutralizing antibody (NAb) responses differ. For the H1 HA antigens, the full-length HA induced significantly higher HI and NAb responses than did the TM-truncated HA. For the H3 HA antigen, both the full-length HA and TM-truncated HA induced high levels of HI and NAb responses. These data indicate that H1 and H3 antigens have different expression requirements for the induction of an optimal protective antibody response and that the structure integrity of HA antigens is critical for eliciting type-specific protective antibody responses. Our findings will have an important impact on future subunit-based flu vaccine development.

Animals↗

PubMed2004

Independent but not synergistic enhancement to the immunogenicity of DNA vaccine expressing HIV-1 gp120 glycoprotein by codon optimization and C3d fusion in a mouse model.

The ability to elicit humoral and cell-mediated immune (CMI) responses from DNA immunization by combinational use of codon optimization and C3d component of complement was evaluated in this study. DNA vaccines that express either the wild type or the codon optimized gp120 gene coding for the envelope (Env) glycoprotein of human immunodeficiency virus (HIV-1) from the primary isolate JR-FL strain were compared to the same forms fused to three tandem copies of the murine C3d genes. Either codon optimization or C3d fusion alone was effective at generating early appearance, higher binding and neutralizing antibody responses. We also observed that cell-mediated immune responses against HIV Env could also be enhanced by C3d fusion. However, for both humoral and CMI responses, there were no synergistic effects when the combination of codon optimization and C3d fusion was employed.

AIDS Vaccines↗

PubMed2006

Novel codon-optimized GM-CSF gene as an adjuvant to enhance the immunity of a DNA vaccine against HIV-1 Gag.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immunomodulatory cytokine. Here we generated a novel codon-optimized murine GM-CSF gene as an adjuvant. The codon-optimized GM-CSF gene significantly increased protein expression levels in all cells tested. Although injection of the wild-type GM-CSF plasmids adjuvanted HIV-1 Gag DNA vaccine induced detectable immune responses, co-administration of plasmids encoding the codon-optimized GM-CSF sequence with the DNA vaccine resulted in a strong antibody and CTL responses and a protective immune response against infection with recombinant vaccinia virus expressing HIV-1 Gag. This novel codon-optimized GM-CSF gene offers a practical molecular strategy for potentiating immune responses to vaccines as well as other immunotherapeutic strategies.

AIDS Vaccines↗

PubMed2001

Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice.

In contrast to conventional vaccines, DNA and other subunit vaccines exclusively utilize host cell molecules for transcription and translation of proteins. The adenine plus thymine content of Plasmodium falciparum gene sequences (approximately 80%) is much greater than that of Homo sapiens (approximately 59%); consequently, codon usage is markedly different. We hypothesized that modifying codon usage of P. falciparum genes encoded by DNA vaccines from that used by the parasite to those resembling mammalian codon usage would lead to increased P. falciparum protein expression in vitro in mouse cells and increased antibody responses in DNA-vaccinated mice. We synthesized gene fragments encoding the receptor-binding domain of the 175-kDa P. falciparum erythrocyte-binding protein (EBA-175 region II) and the 42-kDa C-terminal processed fragment of the P. falciparum merozoite surface protein 1 (MSP-1(42)) using the most frequently occurring codon in mammals to code for each amino acid, and inserted the synthetic genes in DNA vaccine plasmids. In in vitro transient-expression assays, plasmids containing codon-optimized synthetic gene fragments (pS plasmids) showed greater than fourfold increased protein expression in mouse cells compared to those containing native gene fragments (pN plasmids). In mice immunized with 0.5, 5.0, or 50 microg of the DNA plasmids, the dose of DNA required to induce equivalent antibody titers was 10- to 100-fold lower for pS than for pN plasmids. These data demonstrate that optimizing codon usage in DNA vaccines can improve protein expression and consequently the immunogenicity of gene fragments in DNA vaccines for organisms whose codon usage differs substantially from that of mammals.

Animals↗

PubMed1997

Bacterial expression and purification of biologically active mouse c-Fos proteins by selective codon optimization.

A simple strategy using selective codon optimization was devised to express mouse c-Fos protein in high levels in E. coli. Ten arginine codons located in the basic region were optimized to achieve high levels of protein expression. The c-Fos protein was purified to near homogeneity and was demonstrated to be biologically active by assaying its several biological activities.

Animals↗

PubMed2002

eCodonOpt: a systematic computational framework for optimizing codon usage in directed evolution experiments.

We present a systematic computational framework, eCodonOpt, for designing parental DNA sequences for directed evolution experiments through codon usage optimization. Given a set of homologous parental proteins to be recombined at the DNA level, the optimal DNA sequences encoding these proteins are sought for a given diversity objective. We find that the free energy of annealing between the recombining DNA sequences is a much better descriptor of the extent of crossover formation than sequence identity. Three different diversity targets are investigated for the DNA shuffling protocol to showcase the utility of the eCodonOpt framework: (i) maximizing the average number of crossovers per recombined sequence; (ii) minimizing bias in family DNA shuffling so that each of the parental sequence pair contributes a similar number of crossovers to the library; and (iii) maximizing the relative frequency of crossovers in specific structural regions. Each one of these design challenges is formulated as a constrained optimization problem that utilizes 0-1 binary variables as on/off switches to model the selection of different codon choices for each residue position. Computational results suggest that many-fold improvements in the crossover frequency, location and specificity are possible, providing valuable insights for the engineering of directed evolution protocols.

Aldose-Ketose Isomerases↗

PubMed2006

Whole genome analysis of non-optimal codon usage in secretory signal sequences of Streptomyces coelicolor.

Non-optimal (rare) codons have been suggested to reduce translation rate and facilitate secretion in Escherichia coli. In this study, the complete genome analysis of non-optimal codon usage in secretory signal sequences and non-secretory sequences of Streptomyces coelicolor was performed. The result showed that there was a higher proportion of non-optimal codons in secretory signal sequences than in non-secretory sequences. The increased tendency was more obvious when tested with the experimental data of secretory proteins from proteomics analysis. Some non-optimal codons for Arg (AGA, CGU and CGA), Ile (AUA) and Lys (AAA) were significantly over presented in the secretary signal sequences. It may reveal that a balanced non-optimal codon usage was necessary for protein secretion and expression in Streptomyces.

Base Sequence↗