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Results for “codon optimization”

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At least 451 records · Page 25Linked to original sources

PubMed1988

Effect of ribosome binding site on gene expression in Escherichia coli.

Using the expression of human renin gene in Escherichia coli as a model, we have observed that a distance of 7-10 nucleotides between the Shine-Dalgarno site to the initiation codon ATG is optimal for translation initiation. The sequence ATA as the triplet preceding the ATG gives the best expression among those tested. These observations agree with the statistical bias observed for the genes of E. coli and its phages. We have also compared gene expression with three different Shine-Dalgarno sites. Expression of a given gene can be increased by as much as 1000-fold with slight modifications in the Shine-Dalgarno sequence.

Base Sequence↗

PubMed2000

Efficient production of recombinant brazzein, a small, heat-stable, sweet-tasting protein of plant origin.

Brazzein is a 54-amino-acid sweet-tasting protein first isolated from the fruit of Pentadiplandra brazzeana Baillon found in West Africa. Brazzein, as isolated from the fruit, is 500 times sweeter than sucrose on a weight basis (9500 times sweeter on a per-molecule basis). A minor component of brazzein from fruit, des-pGlu1-brazzein, has 53 amino acid residues and has twice the sweetness of the parent protein. We have designed a gene for des-pGlu1- brazzein that incorporates codons that are optimal for protein production in Escherichia coli. Production of brazzein from the chemically synthesized gene resulted in recombinant protein with sweetness similar to that of brazzein isolated from the original source. The best yields were achieved by producing brazzein as a fusion with staphylococcal nuclease with a designed cyanogen bromide cleavage site. Because of its intense sweetness and stability at high pH and temperature, brazzein is an ideal system for investigating the chemical and structural requirements involved in sweet-taste properties. This efficient protein production system for brazzein will facilitate such investigations.

Base Sequence↗

PubMed2003

The Plasmodium falciparum PfGatp is an endoplasmic reticulum membrane protein important for the initial step of malarial glycerolipid synthesis.

During its 48-h asexual life cycle within human erythrocytes, Plasmodium falciparum grows to many times its own size and divides to produce 16-32 new parasites. This rapid multiplication requires active synthesis of new membranes and is fueled by phospholipid precursors and fatty acids that are scavenged from the human host. Plasmodium membrane biogenesis relies heavily on the expression of parasite enzymes that incorporate these precursors into phospholipids. However, little is known about the genes involved in membrane biogenesis or where this process takes place within the parasite. Here, we describe the analysis in P. falciparum of the first step of phospholipid biosynthesis that controls acylation of glycerol 3-phosphate (GPAT) at the sn-1 position. We show that this activity is of parasite origin and is specific for glycerol 3-phosphate substrate. We have identified the gene, PfGAT, encoding this activity in P. falciparum and reconstituted its codon composition for optimal expression in the yeast Saccharomyces cerevisiae. PfGAT complements the lethality of a yeast double mutant gat1Deltagat2Delta, lacking GPAT activity. Biochemical analysis revealed that PfGatp is a low affinity GPAT enzyme with a high specificity for C16:0 and C16:1 substrates. PfGatp is an integral membrane protein of the endoplasmic reticulum expressed throughout the intraerythrocytic life cycle of the parasite but induced mainly at the trophozoite stage. This study, which describes the first protozoan GPAT gene, reveals an important role for the endoplasmic reticulum in the initial step of Plasmodium membrane biogenesis.

Amino Acid Sequence↗

PubMed1994

Molecular characterization of an enterotoxin from Salmonella typhimurium.

In this study, we describe the molecular and antigenic characteristics of a cloned enterotoxin from Salmonella typhimurium strain Q1. The full length Salmonella enterotoxin gene (stn), localized on a 2.8 kb ClaI/PstI DNA fragment, was cloned from a genomic library of Salmonella. Based on nucleotide sequence analysis, the stn gene contained 749 bp that would encode a protein having a molecular size of 29,073. The most unusual feature of the stn gene was the presence of a rare initiation codon (TTG) in lieu of the typical ATG codon, which required site-directed mutagenesis to confirm the precise initiation site. The expression of the stn gene in a bacteriophage T7 RNA polymerase/promoter system was enhanced by introducing a typical ATG start codon and an optimal Shine-Dalgarno sequence upstream of the stn gene by site-directed mutagenesis. The stn gene was located opposite the hydHG operon that regulates labile hydrogenase activity in Salmonella species and Escherichia coli. The overall amino acid sequence of the enterotoxin was quite dissimilar to any other published sequence, including cholera toxin or other adenylate cyclase-activating proteins. However, an intriguing similarity in a small region of the amino acid sequence of Stn was observed with portions of the amino acid sequences from several other protein toxins known to ADP-ribosylate host cell proteins. This region of homology may indicate a conserved motif, within the active site, that is involved in the stimulation of adenylate cyclase activity.

Amino Acid Sequence↗

PubMed2005

Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates.

BACKGROUND: Anemia due to impaired erythropoietin (EPO) production is associated with kidney failure. Recombinant proteins are commonly administered to alleviate the symptoms of this dysfunction, whereas gene therapy approaches envisaging the delivery of EPO genes have been tried in animal models in order to achieve stable and long-lasting EPO protein production. Naked DNA intramuscular injection is a safe approach for gene delivery; however, transduction levels show high inter-individual variability in rodents and very poor efficiency in non-human primates. Transduction can be improved in several animal models by application of electric pulses after DNA injection. METHODS: We have designed a modified EPO gene version by changing the EPO leader sequence and optimizing the gene codon usage. This modified gene was electro-injected into mice, rabbits and cynomolgus monkeys to test for protein production and biological effect. CONCLUSIONS: The modified EPO gene yields higher levels of circulating transgene product and a more significant biological effect than the wild-type gene in all the species tested, thus showing great potential in clinically developable gene therapy approaches for EPO delivery.

Animals↗

PubMed1988

Secretion and export of IGF-1 in Escherichia coli strain JM101.

The processing of LamB-IGF-1 fusion protein and the export of processed IGF-1 (insulin-like growth-factor-1) into the growth medium was examined in the Escherichia coli host strain, JM101. Several strain or plasmid modifications were tried to increase export of periplasmic (processed) IGF-1 into the growth medium of JM101. These included: (1) use of a lon null mutant strain to increase accumulation levels of unprocessed LamB-IGF-1 fusion protein; (2) use of an alternative drug resistance marker on the expression plasmid rather than beta-lactamase, thereby reducing any competition for processing of LamB-IGF-1 by signal peptidase; (3) examination of whether phage M13 gene III protein expression caused more periplasmic IGF-1 to be exported into the growth medium due to increased outer membrane permeability; and (4) examination of the effect of E. coli or yeast optimized IGF-1 codons. None of these strain or plasmid modifications caused any significant increase in export of IGF-1 into the growth medium of JM101. Solubility studies of LamB-IGF-1 and processed IGF-1 showed that virtually all of the LamB-IGF-1 and IGF-1 remaining within the cell after a 2 h induction period was insoluble. This implied that only soluble LamB-IGF-1 was processed to IGF-1 and that only soluble IGF-1 was exported into the growth medium. Taken together, the results indicated that LamB-IGF-1 and IGF-1 solubility were the limiting factors in secretion of IGF-1 into the periplasm and export of IGF-1 into the growth medium.

Amino Acid Sequence↗

PubMed1993

Construction and expression of a synthetic streptavidin-encoding gene in Escherichia coli.

A synthetic gene encoding 'core' streptavidin (SAV) [amino acid (aa) residues 13-140 of Streptomyces avidinii SAV] has been efficiently expressed in Escherichia coli from the IPTG-inducible lac promoter of plasmid pET3a. In this system, expression levels are nearly tenfold greater for the synthetic gene than for the corresponding native gene. The synthetic gene was constructed from overlapping oligodeoxyribonucleotides whose sequences were optimized to incorporate codons preferred by highly expressed E. coli genes. Biochemical characterizations by gel methods, aa analysis, N-terminal sequencing, and size exclusion chromatography show that the synthetic gene product purified by affinity chromatography possesses the properties expected for core SAV.

Amino Acid Sequence↗

PubMed2004

A ROS repressor-mediated binary regulation system for control of gene expression in transgenic plants.

We describe a novel binary system to control transgene expression in plants. The system is based on the prokaryotic repressor, ROS, from Agrobacterium tumefaciens, optimized for plant codon usage and for nuclear targeting (synROS). The ROS protein bound in vitro to double stranded DNA comprising the ROS operator sequence, as well as to single stranded ROS operator DNA sequences, in an orientation-independent manner. A synROS-GUS fusion protein was localized to the nucleus, whereas wtROS-GUS fusion remained in the cytoplasm. The ability of synROS to repress transgene expression was validated in transgenic Arabidopsis thaliana and Brassica napus. When expressed constitutively under the actin2 promoter, synROS repressed the expression of the reporter gene gusA linked to a modified CaMV35S promoter containing ROS operator sequences in the vicinity of the TATA box and downstream of the transcription initiation signal. Repression ranged from 32 to 87% in A. thaliana, and from 23 to 76% in B. napus. These results are discussed in relation to the potential application of synROS in controlling the expression of transgenes and endogenous genes in plants and other organisms.

Agrobacterium tumefaciens↗

PubMed2001

Differential mercury volatilization by tobacco organs expressing a modified bacterial merA gene.

Mercury pollution is a major environmental problem accompanying industrial activities. Most of the mercury released ends up and retained in the soil as complexes of the toxic ionic mercury (Hg2+), which then can be converted by microbes into the even more toxic methylmercury which tends to bioaccumulate. Mercury detoxification of the soil can also occur by microbes converting the ionic mercury into the least toxic metallic mercury (Hg0) form, which then evaporates. The remediation potential of transgenic plants carrying the MerA gene from E. coli encoding mercuric ion reductase could be evaluated. A modified version of the gene, optimized for plant codon preferences (merApe9, Rugh et al. 1996), was introduced into tobacco by Agrobacterium-mediated leaf disk transformation. Transgenic seeds were resistant to HgCl2 at 50 microM, and some of them (10-20% ) could germinate on media containing as much as 350 microM HgCl2, while the control plants were fully inhibited or died on 50 microM HgCl2. The rate of elemental mercury evolution from Hg2+ (added as HgCl2) was 5-8 times higher for transgenic plants than the control. Mercury volatilization by isolated organs standardized for fresh weight was higher (up to 5 times) in the roots than in shoots or the leaves. The data suggest that it is the root system of the transgenic plants that volatilizes most of the reduced mercury (Hg0). It also suggests that much of the mercury need not enter the vascular system to be transported to the leaves for volatilization. Transgenic plants with the merApe9 gene may be used to mercury detoxification for environmental improvement in mercury-contaminated regions more efficiently than it had been predicted based on data on volatilization of whole plants via the upper parts only (Rugh et al. 1996).

Bacterial Proteins↗

PubMed1998

Human glutathione transferase A4-4: an alpha class enzyme with high catalytic efficiency in the conjugation of 4-hydroxynonenal and other genotoxic products of lipid peroxidation.

A sequence encoding a novel glutathione transferase, GST A4-4, has been identified in a human fetal brain cDNA library. The protein has been produced in Escherichia coli after optimization of the codon usage for high-level heterologous expression. The dimeric protein has a subunit molecular mass of 25704 Da based on the deduced amino acid composition. Human GST A4-4 is a member of the Alpha class but shows only 53% amino acid sequence identity with the major liver enzyme GST A1-1. High catalytic efficiency with 4-hydroxyalkenals and other cytotoxic and mutagenic products of radical reactions and lipid peroxidation is a significant feature of GST A4-4. The kcat/Km values for 4-hydroxynonenal and 4-hydroxydecenal are > 3 x 10(6) M-1. s-1, several orders of magnitude higher than the values for conventional GST substrates. 4-Hydroxynonenal and other reactive electrophiles produced by oxidative metabolism have been linked to aging, atherosclerosis, cataract formation, Parkinson's disease and Alzheimer's disease, as well as other degenerative human conditions, suggesting that human GST A4-4 fulfills an important protective role and that variations in its expression may have significant pathophysiological consequences.

Aldehydes↗

PubMed2000

Monitoring phase-specific gene expression in Histoplasma capsulatum with telomeric GFP fusion plasmids.

Dimorphism is an essential feature of Histoplasma capsulatum pathogenesis, and much attention has been focused on characteristics that are unique to the saprophytic mycelial phase or the parasitic yeast phase. Recently, we identified a secreted calcium-binding protein, CBP, that is produced in large amounts by yeast cells but is undetectable in mycelial cultures. In this study, the green fluorescent protein (GFP) was established as a reporter in H. capsulatum to study regulation of CBP1 expression in cultures and in single cells grown under different conditions and inside macrophages. One GFP version that was optimized for human codon usage yielded highly fluorescent Histoplasma yeast cells. By monitoring GFP fluorescence during the transition from mycelia to yeast, we demonstrated that the CBP1 promoter is only fully active after complete morphological conversion to the yeast form, indicating for the first time that CBP1 is developmentally regulated rather than simply temperature regulated. Continuous activity of the CBP1 promoter during infection of macrophages supports the hypothesis that CBP secretion plays an important role for Histoplasma survival within the phagolysosome. Broth cultures of Histoplasma yeasts carrying a CBP-GFP protein fusion construct were able to secrete a full-length fluorescent fusion protein that remained localized within the phagolysosomes of infected macrophages. Additionally, a comparison of two Histoplasma strains carrying the CBP1 promoter fusion construct either epichromosomally or integrated into the chromosome revealed cell-to-cell variation in plasmid copy number due to uneven plasmid partitioning into daughter cells.

Animals↗

PubMed2000

Rev-independent expression of synthetic gag-pol genes of human immunodeficiency virus type 1 and simian immunodeficiency virus: implications for the safety of lentiviral vectors.

The safety of lentiviral vectors for clinical applications is still a major concern. The gag-pol expression plasmids and the lentiviral vectors used in previous studies contain homologous regions, which constitute a risk for recombination events. Synthetic gag-pol genes of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were therefore constructed, in which the codon usage was optimized for expression in human cells without altering the amino acid sequences. The synthetic gag-pol genes allowed efficient expression of these genes in the absence of Rev and the 5' untranslated leader region. Both the HIV-1 and the SIV synthetic gag-pol expression plasmids could mediate transduction of an SIV vector into nondividing human cells with titers of about 10(6) transducing units/ml. Similar titers were obtained with a four-plasmid vector-packaging system based on HIV-1. Using a biological assay, homologous recombination events between the synthetic gag-pol expression plasmids and an SIV vector were undetectable and in comparison with a previously used gag-pol expression plasmid at least approximately 100-fold less frequent. By eliminating regions of homology and sequences involved in packaging, synthetic gag-pol genes should improve the safety profile of lentiviral vectors.

5' Untranslated Regions↗

PubMed1997

Expression of natural and synthetic genes encoding herpes simplex virus 1 protease in Escherichia coli and purification of the protein.

An attractive target for anti-herpes chemotherapy is the herpes simplex virus 1 (HSV-1) protease encoded by the UL26 gene. Studies with HSV-1 strains that harbour mutations in the protease gene have demonstrated that the protease is essential for DNA packaging and virus maturation. The UL26 translation product is 635 amino acids long and undergoes autoproteolytic processing between residues Ala247/Ser248 and Ala610/Ser611. The N-terminal processing product (amino acids 1-247) contains the protease domain. To perform crystallization studies and high throughput screening for potent inhibitors, large amounts of the HSV-1 protease are required. However, expression of the natural HSV-1 protease gene in Escherichia coli using a T7-promoter-regulated system is low and does not allow for the efficient production of larger amounts of highly purified enzyme. In this report, we describe the use of a synthetic protease gene with optimized E. coli codon usage. The level of protease expression was at least 20 times higher with the synthetic gene as compared to the natural UL26 gene. The HSV-1 protease was purified to homogeneity in three steps using mixed-bed ion-exchange chromatography, affinity chromatography, and hydroxyapatite chromatography.

Amino Acid Sequence↗

PubMed1996

Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains.

A study was carried out to obtain more insight into the parameters that determine the secretion of heterologous proteins from filamentous fungi. A strategy was chosen in which the mRNA levels and protein levels of a number of heterologous genes of different origins were compared. All genes were under control of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) expression signals and were integrated in a single copy at the A. awamori pyrG locus. A Northern (RNA) analysis showed that large differences occurred in the steady-state mRNA levels obtained with the various genes; those levels varied from high values for genes of fungal origin (A. awamori 1,4-beta-endoxylanase A, Aspergillus niger glucoamylase, and Thermomyces lanuginosa lipase) to low values for genes of nonfungal origin (human interleukin 6 and Cyamopsis tetragonoloba [guar] alpha-galactosidase). With the C. tetragonoloba alpha-galactosidase wild-type gene full-length mRNA was even undetectable. Surprisingly, small amounts of full-length mRNA could be detected when a C. tetragonoloba alpha-galactosidase gene with an optimized Saccharomyces cerevisiae codon preference was expressed. In all cases except human interleukin 6, the protein levels corresponded to the amounts expected on basis of the mRNA levels. For human interleukin 6, very low protein levels were observed, whereas relatively high steady-state mRNA levels were obtained. Our data suggest that intracellular protein degradation is the most likely explanation for the low levels of secreted human interleukin 6.

Aspergillus↗

PubMed1997

Glucoamylase gene fusions alleviate limitations for protein production in Aspergillus awamori at the transcriptional and (post) translational levels.

In this study we have analyzed the effects of a glucoamylase gene fusion on the mRNA levels and protein levels for the human interleukin-6 gene (hil6) and the guar alpha-galactosidase gene (aglA). Previously it was shown that production of nonfused alpha-galactosidase and hIL-6 in Aspergillus awamori was limited at transcriptional and (post)translational levels, respectively (R. J. Gouka, P. J. Punt, J. G. M. Hessing, and C. A. M. J. J. van den Hondel, Appl. Environ. Microbiol. 62:1951-1957, 1996). Vectors were constructed which contained either the hil6 or aglA gene fused to the Aspergillus niger glucoamylase gene (glaA) under control of the efficient 1,4-beta-endoxylanase A promoter and transcription terminator. For comparison, the vectors were integrated in a single copy at the pyrG locus of A. awamori. A glaA fusion to the 5' end of the hil6 gene resulted in a large increase in hIL-6 yield, whereas with a glaA fusion to the 3' end of the hil6 gene, almost no protein was produced. Nevertheless, the steady-state mRNA levels of both fusions were very similar and not clearly increased compared to those of a strain expressing nonfused hIL-6. Fusions of glaA to the 5' end of the wild-type guar aglA gene resulted in truncated mRNA lacking almost 900 bases (> 80%) of the aglA sequence. When the coding sequence of the wild-type aglA gene was replaced by a synthetic aglA gene with optimized Saccharomyces cerevisiae codon usage, full-length mRNA was obtained. Compared to a nonfused synthetic aglA gene, a glaA fusion with the synthetic aglA gene resulted in a 25-fold increase in the mRNA level and, as a consequence, a similar increase in the alpha-galactosidase protein level. The truncated transcripts derived from the wild-type aglA gene were further analyzed by nuclear run-on transcription assays. These experiments indicated that transcription elongation in the nucleus proceeded at least 400 bases downstream of the site where the truncation was determined, indicating that transcription elongation or premature termination was not the reason for the generation of truncated mRNAs. As the truncated mRNA also contained a poly(A) tail, truncation most likely occurs by incorrect processing of the aglA mRNA in the nucleus.

Amino Acid Sequence↗

PubMed2004

Cryptococcus neoformans virulence gene discovery through insertional mutagenesis.

Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

Acetyltransferases↗

PubMed1993

Cloning and expression of a member of a new cytochrome P-450 family: cytochrome P-450lin (CYP111) from Pseudomonas incognita.

Cytochrome P-450lin catalyzes the 8-methyl hydroxylation of linalool as the first committed step of its utilization by Pseudomonas incognita as the sole carbon source. By using a polymerase chain reaction-based cloning strategy, a 2.1-kb DNA fragment containing the cytochrome P-450lin gene (linC) was isolated. An open reading frame of 406 amino acids has been identified as that of P-450lin on the basis of amino acid sequence data from peptides of the native protein. Heterologous expression of functional holoprotein is exhibited by Escherichia coli transformed with pUC18 containing the subcloned linC gene under constitutive transcriptional control of the lac promoter. The G+C content of linC was found to be 55% overall and 58% in the third codon position. An optimized amino acid sequence alignment of P-450lin with cytochrome P-450cam shows that the two enzymes have only 25% identity. P-450lin was found to exhibit the expected conservation in the axial cysteine heme ligand-containing peptide and the threonine region postulated to form an O2-binding pocket (T. L. Poulos, B. C. Finzel, and A. J. Howard, J. Mol. Biol. 195:687-700, 1987). The low amino acid sequence identity between P-450lin and all other P-450 sequences has shown that P-450lin is the first member of the CYP111 P-450 gene family.

Amino Acid Sequence↗

PubMed2004

Molecular cloning of a gene encoding endo-beta-D-1,4-glucanase PCE1 from Phycomyces nitens.

We previously cloned three endoglucanase genes, rce1, rce2, and rce3, from Rhizopus oryzae as the first cellulase genes from the subdivision Zygomycota. In this study, an endoglucanase gene, designated a pce1 gene, was cloned by plaque hybridization with the codon usage-optimized rce1 gene as a probe from Phycomyces nitens, a member of the subdivision Zygomycota. The pec1 gene had an open reading frame of 1,038 nucleotides encoding an endoglucanase (PCE1) of 346 amino acid residues. The amino acid sequence deduced from the pce1 gene consisted of a cellulose-binding domain (CBD) at the N terminus and of a catalytic domain belonging to family 45 glycoside hydrolase at the C terminus. PCE1 was purified to apparent homogeneity from the culture supernatant of P. nitens and the molecular mass was found to be 45 kDa. The optimum pH for the CMCase activity of PCE1 was 6.0, and the optimum temperature was 50 degrees C, the lowest among the family 45 endoglucanases.

Amino Acid Sequence↗