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At least 433 records · Page 24Linked to original sources
PubMed2003

Optimized gene synthesis and high expression of human interleukin-18.

Human interleukin-18 (hIL-18), originally known as an IFN-gamma-inducing factor, is a recently cloned cytokine that is secreted by Kupffer cells of the liver and by stimulated macrophages. We have previously established a method of expression and purification of IL-18. The yield however remains low and the insufficient expression of a heterologous protein could be due to skewed codon usage between the expression host and the cDNA donor. The sequence of mature hIL-18 has 37 a.a. rare codons for Escherichia coli in a total of 157 a.a. To overcome this problem, gene synthesis was performed with optimized codons for the expression host E. coli. The final yield of the hIL-18 protein with optimized codons was about five times higher than the yield with the native sequence. Using a minimal medium, this system produces large quantities of labeled proteins that can be used in NMR analysis. Our simple and efficient production system can be applied to the production of other cytokines for new structural and therapeutic use.

Amino Acid Sequence↗
PubMed2006

Optimized FaeG expression and a thermolabile enterotoxin DNA adjuvant enhance priming of an intestinal immune response by an FaeG DNA vaccine in pigs.

One of the problems hindering the development of DNA vaccines is the relatively low immunogenicity often seen in humans and large animals compared to that in mice. In the present study, we tried to enhance the immunogenicity of a pcDNA1/faeG19 DNA vaccine in pigs by optimizing the FaeG expression plasmid and by coadministration of the plasmid vectors encoding the A and B subunits of the Escherichia coli thermolabile enterotoxin (LT). The insertion of a Kozak sequence and optimization of vector (cellular localization and expression) and both vector and codon usage were all shown to enhance in vitro FaeG expression compared to that of pcDNA1/faeG19. Subsequently, pcDNA1/faeG19 and the vector-optimized and the vector-codon-optimized construct were tested for their immunogenicity in pigs. In line with the in vitro results, antibody responses were better induced with increasing expression. The LT vectors additionally enhanced the antibody response, although not significantly, and were necessary to induce an F4-specific cellular response. These vectors were also added because LT has been described to direct the systemic response towards a mucosal immunoglobulin A (IgA) response in mice. Here, however, the intradermal FaeG DNA prime-oral F4 boost immunization resulted in a mainly systemic IgG response, with only a marginal but significant reduction in F4+ E. coli fecal excretion when the piglets were primed with pWRGFaeGopt and pWRGFaeGopt with the LT vectors.

Adhesins, Escherichia coli↗
PubMed1997

Does Escherichia coli optimize the economics of the translation process?

The codon translation rate is usually assumed to be proportional to the cellular concentration of the cognate tRNA, but synonymous codons sharing the same cognate tRNA may be translated at rather different rates. To account for the latter observation, we assume that the translation process is optimized in two respects: (i), the codon demand is optimized with respect to the supply of cognate tRNAs (composition of the tRNA pool); and (ii), for synonymous codons sharing the same cognate tRNA, the usage frequency of each codon correlates optimally with the stability of the codon-anticodon complex. These assumptions allow us to compute the relative rate constants of synonymous codons. Highly expressed genes, which produce 80-90% of the protein mass in the E. coli cell, appear to have selected codons which make an optimal use of the tRNA pool. Assuming the optimization criteria were valid, a list of codon translation times (in ms) were derived from available experimental data.

Codon, Terminator↗
PubMed1987

High-level production of fully active human alpha 1-antitrypsin in Escherichia coli.

The human alpha-1-antitrypsin (A1AT) gene expressed in Escherichia coli as a full-length, non-fusion gene product accumulates to a relatively low level approaching less than or equal to 0.1% of total cellular protein. In contrast, deletion of the first 5, 10 or 15 codons leads to production of truncated A1AT derivatives at levels between 10 and 30% of total cellular protein. The protein with the largest truncation was insoluble and inactive following solubilization by chaotropic agents. In contrast, the two derivatives with the smaller truncations were found to be soluble, and exhibit identical specific activities in both trypsin and elastase inhibition assays to authentic human A1AT. The expression of the full-length A1AT was also optimized by making silent third position mutations within its first 15 codons. These mutations were chosen to optimize codon usage and minimize the possibility of RNA secondary structure formation in this region. Via this approach, expression of full-length, authentic, fully active A1AT was increased at least 20-fold to 2% of total cellular protein. Optimal expression was obtained using as few as three silent mutations in the first five codons, confirming the importance of this 5'-terminal region as had been defined by our deletion mutants. Both the full-length derivatives as well as the small N-terminal deletion derivative can be readily purified from bacterial extracts in fully active form suitable for the examination of their potential therapeutic application.

Base Sequence↗
PubMed2000

Studies on codon usage in Entamoeba histolytica.

Codon usage bias of Entamoeba histolytica, a protozoan parasite, was investigated using the available DNA sequence data. Entamoeba histolytica having AT rich genome, is expected to have A and/or T at the third position of codons. Overall codon usage data analysis indicates that A and/or T ending codons are strongly biased in the coding region of this organism. However, multivariate statistical analysis suggests that there is a single major trend in codon usage variation among the genes. The genes which are supposed to be highly expressed are clustered at one end, while the majority of the putatively lowly expressed genes are clustered at the other end. The codon usage pattern is distinctly different in these two sets of genes. C ending codons are significantly higher in the putatively highly expressed genes suggesting that C ending codons are translationally optimal in this organism. In the putatively lowly expressed genes A and/or T ending codons are predominant, which suggests that compositional constraints are playing the major role in shaping codon usage variation among the lowly expressed genes. These results suggest that both mutational bias and translational selection are operational in the codon usage variation in this organism.

Animals↗
PubMed2002

The influence of viral coding sequences on pestivirus IRES activity reveals further parallels with translation initiation in prokaryotes.

Classical swine fever virus (CSFV) is a member of the pestivirus family, which shares many features in common with hepatitis C virus (HCV). It is shown here that CSFV has an exceptionally efficient cis-acting internal ribosome entry segment (IRES), which, like that of HCV, is strongly influenced by the sequences immediately downstream of the initiation codon, and is optimal with viral coding sequences in this position. Constructs that retained 17 or more codons of viral coding sequence exhibited full IRES activity, but with only 12 codons, activity was approximately 66% of maximum in vitro (though close to maximum in transfected BHK cells), whereas with just 3 codons or fewer, the activity was only approximately 15% of maximum. The minimal coding region elements required for high activity were exchanged between HCV and CSFV. Although maximum activity was observed in each case with the homologous combination of coding region and 5' UTR, the heterologous combinations were sufficiently active to rule out a highly specific functional interplay between the 5' UTR and coding sequences. On the other hand, inversion of the coding sequences resulted in low IRES activity, particularly with the HCV coding sequences. RNA structure probing showed that the efficiency of internal initiation of these chimeric constructs correlated most closely with the degree of single-strandedness of the region around and immediately downstream of the initiation codon. The low activity IRESs could not be rescued by addition of supplementary eIF4A (the initiation factor with ATP-dependent RNA helicase activity). The extreme sensitivity to secondary structure around the initiation codon is likely to be due to the fact that the eIF4F complex (which has eIF4A as one of its subunits) is not required for and does not participate in initiation on these IRESs.

5' Untranslated Regions↗
PubMed1990

Codon usage and secondary structure of mRNA.

The specific codon usage pattern of the repetitive unit nucleotide sequence of silk fibroin mRNA suggests that selection has operated on the codon usage to optimize the secondary structure characteristic of the mRNA. The correlation between the stability map of local secondary structure of type I collagen mRNA and the codon usage pattern and the translation rate of the collagen is also implied.

Animals↗
PubMedDate not supplied

[Non-canonical decoding events at stop codons in eukaryotes].

Regulation of protein synthesis at translation termination is a relatively under-explored, but rapidly expanding field. Recent advances in elucidating the mechanism of translation termination are helping to understand non-canonical events associated with translation termination. These "recoding" events include read-through of stop-codons, insertion of unusual amino acids such as selenocysteine and production of several polypeptides from one open reading frame. This review summarises data on termination-dependent recoding events, and proposes that there are two types of stop codon-associated sequences optimized to perform different functions: termination of translation per se or alternative elongation events.

Animals↗
PubMed1998

How optimized is the translational machinery in Escherichia coli, Salmonella typhimurium and Saccharomyces cerevisiae?

The optimization of the translational machinery in cells requires the mutual adaptation of codon usage and tRNA concentration, and the adaptation of tRNA concentration to amino acid usage. Two predictions were derived based on a simple deterministic model of translation which assumes that elongation of the peptide chain is rate-limiting. The highest translational efficiency is achieved when the codon recognized by the most abundant tRNA reaches the maximum frequency. For each codon family, the tRNA concentration is optimally adapted to codon usage when the concentration of different tRNA species matches the square-root of the frequency of their corresponding synonymous codons. When tRNA concentration and codon usage are well adapted to each other, the optimal content of all tRNA species carrying the same amino acid should match the square-root of the frequency of the amino acid. These predictions are examined against empirical data from Escherichia coli, Salmonella typhimurium, and Saccharomyces cerevisiae.

Codon↗
PubMed1988

Specific codon usage pattern and its implications on the secondary structure of silk fibroin mRNA.

We have identified two distinctive regions of the repetitive unit nucleotide sequence of fibroin mRNA of Bombyx mori. The codon usage for the major amino acids, glycine, alanine and serine is distinctly different in these two regions, indicating that it is determined by the fibroin mRNA or gene structure but not by the tRNA population. Comparative computer analyses of nucleotide substitutions in the unit sequence suggest that selection has operated on the codon usage to optimize the secondary structure characteristic of the fibroin mRNA.

Amino Acid Sequence↗
PubMed2005

Mass spectrometry of the M. smegmatis proteome: protein expression levels correlate with function, operons, and codon bias.

The fast-growing bacterium Mycobacterium smegmatis is a model mycobacterial system, a nonpathogenic soil bacterium that nonetheless shares many features with the pathogenic Mycobacterium tuberculosis, the causative agent of tuberculosis. The study of M. smegmatis is expected to shed light on mechanisms of mycobacterial growth and complex lipid metabolism, and provides a tractable system for antimycobacterial drug development. Although the M. smegmatis genome sequence is not yet completed, we used multidimensional chromatography and tandem mass spectrometry, in combination with the partially completed genome sequence, to detect and identify a total of 901 distinct proteins from M. smegmatis over the course of 25 growth conditions, providing experimental annotation for many predicted genes with an approximately 5% false-positive identification rate. We observed numerous proteins involved in energy production (9.8% of expressed proteins), protein translation (8.7%), and lipid biosynthesis (5.4%); 33% of the 901 proteins are of unknown function. Protein expression levels were estimated from the number of observations of each protein, allowing measurement of differential expression of complete operons, and the comparison of the stationary and exponential phase proteomes. Expression levels are correlated with proteins' codon biases and mRNA expression levels, as measured by comparison with codon adaptation indices, principle component analysis of codon frequencies, and DNA microarray data. This observation is consistent with notions that either (1) prokaryotic protein expression levels are largely preset by codon choice, or (2) codon choice is optimized for consistency with average expression levels regardless of the mechanism of regulating expression.

Bacterial Proteins↗
PubMed1995

Translational inhibition by a human cytomegalovirus upstream open reading frame despite inefficient utilization of its AUG codon.

The second of three short upstream open reading frames (uORF2) in the transcript leader of the human cytomegalovirus gp48 (gpUL4) virion glycoprotein gene inhibits downstream translation approximately 10-fold. Remarkably, this inhibition depends on the amino acid coding information of uORF2. In the current studies we demonstrate that expression of the cistron downstream from uORF2 depends on ribosomes bypassing the uORF2 AUG codon (AUG2) by a leaky scanning mechanism. Replacing the nucleotides surrounding the wild-type AUG2 codon with those optimal for translation initiation reduces downstream translation approximately 10-fold. Analyses of mutants in which uORF2 either overlaps or is in frame with the downstream reading frame reveal that the initiation frequency at the wild-type AUG2 codon is surprisingly low; rather, the majority of ribosomal subunits bypass the wild-type AUG2 codon because of its suboptimal context. We propose a model to explain this unprecedented example of a paradoxically strong inhibitory effect of an upstream ORF despite inefficient utilization of its initiation codon.

Base Sequence↗
PubMed1998

High-level expression of rat farnesyl:protein transferase in Escherichia coli as a translationally coupled heterodimer.

Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid group from farnesyl diphosphate to the CaaX cysteine of a variety of cellular proteins. Since FPTase is a large (95-kDa) heterodimeric protein and is inactive unless the alpha- and beta-subunits are coexpressed, large-scale overexpression of active enzyme has been challenging. We report the design of a translationally coupled expression system that will produce FPTase at levels as high as 30 mg/L Escherichia coli. Heterodimeric expression of FPTase was achieved using a translationally coupled operon from the T7 promoter of the pET23a (Novagen) expression plasmid. The beta-subunit-coding sequence was placed upstream of the alpha-subunit coding sequence linked by overlapping beta-subunit stop and alpha-subunit start codons. Additionally, the initial 88 codons of the alpha-subunit gene were altered, removing rare codons and replacing them with codons used in highly expressed proteins in E. coli. Since previous attempts at recombinantly expressing FPTase in E. coli from a translationally coupled system have demonstrated that initiation of translation of the alpha-subunit is poor, we propose that the optimization of the codons at the start of the alpha-subunit gene leads to the observed high level of recombinant expression.

Alkyl and Aryl Transferases↗
PubMed2006

Prokaryotes that grow optimally in acid have purine-poor codons in long open reading frames.

In nucleic acids the N-glycosyl bonds between purines and their ribose sugar moities are broken under acid conditions. If one strand of a duplex DNA segment were more vulnerable to mutation than the other, then the archaeon Picrophilus torridus, with an optimum growth pH near zero, could have adapted by decreasing the purine content of that strand. Yet, P. torridus has an optimum growth temperature near 60 degrees C, and thermophiles prefer purine-rich codons. We found that, as in other thermophiles, high growth temperature correlates with the use of purine-rich codons. The extra purines are often in third, non-amino acid determining, codon positions. However, as in other acidophiles, as open reading frame lengths increase, there is increased use of purine-poor codons, particularly those without purines in second, amino acid-determining, codon positions. Thus, P. torridus can be seen as adapting (a) to temperature by increasing its purines in all open reading frames without greatly impacting protein amino acid compositions, and (b) to pH by decreasing purines in longer open reading frames, thereby potentially impacting protein amino acid compositions. It is proposed that longer open reading frames, being larger mutational targets, have become less vulnerable to depurination by virtue of pyrimidine for purine substitutions.

Adaptation, Physiological↗
PubMed2005

Improved heterologous gene expression in Escherichia coli by optimization of the AT-content of codons immediately downstream of the initiation codon.

Escherichia coli is the most frequently used host for heterologous gene expression. This study focuses on the effect of AT-rich codons immediately downstream of the initiation codon of the target gene. The third to sixth codons of ndx3, a Nudix hydrolase gene from Thermus thermophilus HB8, were engineered by introducing several silent mutations. As a result, the expression level of ndx3 increased in proportion to the AT-content in the third to sixth codons. This result suggests that incorporation of AT-rich codons can be utilized as a general strategy for improving the expression efficiency of a recombinant protein.

AT Rich Sequence↗
PubMed1990

Growth rate dependence of transfer RNA abundance in Escherichia coli.

We have tested the predictions of a model that accounts for the codon preferences of bacteria in terms of a growth maximization strategy. According to this model the tRNA species cognate to minor and major codons should be regulated differently under different growth conditions: the isoacceptors cognate to major codons should increase at fast growth rates while those cognate to minor codons should decrease at fast growth rates. We have used a quantitative Northern blotting technique to measure the abundance of the methionine and the leucine isoacceptor families over growth rates ranging from 0.5 to 2.1 doublings per hour. Five tRNA species that are cognate to major codons (tRNA(eMet), tRNA(1fMet), tRNA(2fMet), tRNA(1Leu) and tRNA(3Leu) increase both as a relative fraction of total tRNA and in absolute concentration with increasing growth rates. Three tRNA species that are cognate to minor codons (tRNA(2Leu), tRNA(4Leu) and tRNA(5Leu) decrease as a relative fraction of total RNA and in absolute concentration with increasing growth rates. These data suggest that the abundances of groups of tRNA species are regulated in different ways, and that they are not regulated simply according to isoacceptor specificity. In particular, the data support the growth optimization model for codon bias.

Base Sequence↗
PubMed1994

Expression of chicken linker histones in E. coli: sources of problems and methods for overcoming some of the difficulties.

Expression of histones in Escherichia coli is important in structural studies on chromatin, because it allows isotopic labeling such as deuteration and replacement of methionines with selenomethionine as well as expression of specific domains of histones. We show that full-length H5 cannot be expressed in E. coli. We have determined that the problem is translational rather than transcriptional. Pulse-labeling studies show that protein turnover is not the reason for lack of accumulation. On dissecting the gene, we find that the problem lies in expressing the highly charged C-terminal tail of H5. We can make progressively increasing amounts of the tail, but at the point where over two-thirds of this region is transcribed, the protein ceases to be made. Surprisingly, full-length H1 is made. In vitro studies show that the H5 gene can be translated in a rabbit reticulocyte system but not in an E. coli system, suggesting that there may be a difference in the ability of eukaryotic and prokaryotic ribosomes to translate this message. The expression of the globular domains of H5 and H1 posed a different problem. There was little or no expression of some of the constructs, even though they were fragments of larger constructs that were well made. Replacement of the first five codons downstream of the initiating ATG codon with those optimized for E. coli, and which were AT rich, restored expression. This may have general implications for expression of eukaryotic proteins in E. coli.

Amino Acid Sequence↗