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PubMed2004

Optimization of codon pair use within the (GGGGS)3 linker sequence results in enhanced protein expression.

Here, we report that a significant increase in recombinant fusion antibody expression can be accomplished by adjusting the nucleotide sequence to conform to certain codon pairing rules. We investigated the expression of a protein in which a single chain Fv specific for HER2/neu with VH and VL joined by a flexible (GGGGS)3 linker was linked to the CH3 of a human anti-rat transferrin receptor IgG3 heavy chain with the same flexible (GGGGS)3 linker. In initial experiments we failed to achieve significant expression of this protein. However, when we made a single nucleotide change in each (GGGGS)3 linker we were able to achieve expression The change of one nucleotide within each linker did not alter either the amino acid sequence or the frequency score of these codon triplets' usage in mammalian cells. Instead they removed two codon pairs predicted to be detrimental to expression. In a transient transfection assay we find that this change results in an over 30-fold increase in expression that is not the result of an increase in the level of accumulated mRNA. In addition, the changes made it possible to isolate stably transfected mammalian cell clones producing high levels of fusion protein, which had not been possible using the original gene.

Amino Acid Sequence↗
PubMed2005

Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris.

Anti-ErbB2 antibodies are used as convenient tools in exploration of ErbB2 functional mechanisms and in treatment of ErbB2-overexpressing tumors. When we employed the yeast Pichia pastoris to express an anti-ErbB2 single-chain antibody (scFv) derived from the tumor-inhibitory monoclonal antibody A21, the yield did not exceed 1-2 mg/L in shake flask cultures. As we considered that the poor codon usage bias may be one limiting factor leading to the inefficient translation and scFv production, we designed and synthesized the full-length scFv gene by choosing the P. pastoris preferred codons while keeping the G+C content at relatively low level. Codon optimization increased the scFv expression level 3- to 5-fold and up to 6-10 mg/L. Northern blotting further confirmed that the increase of scFv expression was mainly due to the enhancement of translation efficiency. Investigation of culture conditions revealed that the maximal cell growth and scFv expression were achieved at pH 6.5-7.0 with 2% casamino acids after 72 h methanol induction. Secreted scFv was easily purified (>95% homogeneous product) from culture supernatants in one step by using Ni2+ chelating affinity chromatography. The yield was approximately 10-15 mg/L. Functional studies showed that the A21 scFv could be internalized with high efficiency after binding to the ErbB2-overexpressing cells, suggesting this regent may prove especially useful for ErbB2-targeted immunotherapy.

Amino Acid Sequence↗
PubMed2006

Long stretches of sequential and identical serine or alanine codons are compatible with an efficient full-length protein expression in Escherichia coli.

The Schistosoma japonicum glutathione S-transferase (GST) recombinant cDNAs, carrying blocks of sequential and identical triplets, consisting of 15-30-45 GCT (Ala) codons or 15-30 and also up to 75 AGC (Ser) codons, are expressed efficiently in an Escherichia coli system in the form of full-length protein chains, as detected by Coomassie-stained SDS-polyacrylamide gels, and soluble fusion proteins are purified by GSH-affinity chromatography. High expression levels and high yields of purified recombinant proteins are achieved. The efficient protein expression is independent of the molecular context and position of the polySer/polyAla string inserted into the GST carrier (near the part of the gene encoding the N- or the C-terminus). These findings suggest that E. coli is a powerful biological system to express foreign genes carrying long stretches coding for Ser- or Ala-rich domains, which are not uncommon in eukaryotic proteins. Moreover, data reported here show that the negative effect of sequential serine codons on protein expression in bacteria, previously reported in the literature, is not a general phenomenon.

Alanine↗
PubMed2005

Characterization of the Micromonospora rosaria pMR2 plasmid and development of a high G+C codon optimized integrase for site-specific integration.

pMR2, an 11.1 kb plasmid was isolated from Micromonospora rosaria SCC2095, NRRL3718, and its complete nucleotide sequence determined. Analysis revealed 13 ORFs including homologs of a KorSA regulatory protein and TraB plasmid transfer protein found on other actinomycete plasmids. pMR2 contains att/int functions consisting of an integrase, an excisionase, and a putative plasmid attachment site (attP). The integrase gene contained a high frequency of codons rarely used in high G+C actinomycete coding regions. The gene was codon optimized for actinomycete codon usage to create the synthetic gene int-OPT. pSPRX740, containing an rpsL promoter and the att/int-OPT region, was introduced into Micromonospora halophytica var. nigra ATCC33088. Analysis of DNA flanking the pSPRX740 integration site confirmed site-specific integration into a tRNA(Phe) gene in the M. halopytica var. nigra chromosome. The pMR2 attP element and chromosomal attachment (attB) site contain a 63 bp region of sequence identity overlapping the 3' end of the tRNA(Phe) gene. Plasmids comprising the site-specific att/int-OPT functions of pMR2 can be used to integrate genes into the chromosome of actinomycetes with an appropriate tRNA gene. The development of an integrative system for Micromonospora will expand our ability to study antibiotic biosynthesis in this important actinomycete genus.

Attachment Sites, Microbiological↗
PubMed2006

Regulated translational bypass of stop codons in yeast.

Stop codons are used to signal the ribosome to terminate the decoding of an mRNA template. Recent studies on translation termination in the yeast Saccharomyces cerevisiae have not only enabled the identification of the key components of the termination machinery, but have also revealed several regulatory mechanisms that might enable the controlled synthesis of C-terminally extended polypeptides via stop-codon readthrough. These include both genetic and epigenetic mechanisms. Rather than being a translation 'error', stop-codon readthrough can have important effects on other cellular processes such as mRNA degradation and, in some cases, can confer a beneficial phenotype to the cell.

Codon, Terminator↗
PubMed1993

Clinical features of autosomal dominant retinitis pigmentosa with rhodopsin gene codon 17 mutation and retinal neovascularization in a Japanese patient.

A 49-year-old Japanese man had autosomal dominant retinitis pigmentosa with a point mutation in codon 17 of the rhodopsin gene, resulting in a threonine-to-methionine change, and retinal neovascularization in both eyes. Pigmentary degeneration mainly in the inferior area of the fundus, and severe loss in the upper portion of the visual field were observed. Moderately preserved rod and cone functions were demonstrated by electroretinograms. These findings differed from those of Japanese and white patients with autosomal dominant retinitis pigmentosa with a codon 347 mutation and were almost the same as those of white patients with the codon 17 mutation. Our study indicates that phenotypic similarities exist among patients with the same mutation, but of different racial backgrounds. The neovascularization in the right eye diminished over a two-year period in conjunction with the progression of retinal degeneration.

Adaptation, Ocular↗
PubMed2003

Seven GC-rich microbial genomes adopt similar codon usage patterns regardless of their phylogenetic lineages.

Seven GC-rich (group I) and three AT-rich (group II) microbial genomes are analyzed in this paper. The seven microbes in group I belong to different phylogenetic lineages, even different domains of life. The common feature is that they are highly GC-rich organisms, with more than 60% genomic GC content. Group II includes three bacteria, which belong to the same subdivision as Pseudomonas aeruginosa in group I. The genomic GC content of the three bacteria is in the range of 26-50%. It is shown that although the phylogenetic lineages of the organisms in group I are remote, the common feature of highly genomic GC content forces them to adopt similar codon usage patterns, which constitutes the basis of an algorithm using a set of universal parameters to recognize known genes in the seven genomes. The common codon usage pattern of function known genes in the seven genomes is GGS type, where G, G, and S are the bases of G, non-G, and G/C, respectively. On the contrary, although the phylogenetic lineages of the three bacteria in group II are quite close, the codon usage patterns of function known genes in these genomes are obviously distinct. There are no universal parameters to identify known genes in the three genomes in group II. It can be deduced that the genomic GC content is more important than phylogenetic lineage in gene recognition programs. We hope that the work might be useful for understanding the common characteristics in the organization of microbial genomes.

Algorithms↗
PubMed2003

A mutation at the start codon defines the differential requirement of dpy-11 in Caenorhabditis elegans body hypodermis and male tail.

dpy-11 encodes a thioredoxin-like molecule that is important for both body and male sensory ray morphogenesis in Caenorhabditis elegans. A mutant allele, s287, has a point mutation with its start codon, AUG, converted into AUA, presumably leading to null function. Since only a weak loss-of-function phenotype was observed, we tested whether an alternative start codon or the converted AUA could be used for translation initiation with reduced efficiency. Based on a functional assay of mutant phenotype complementation and biochemical analysis examining the in vivo synthesis of wild-type and mutant proteins, we conclude that AUA can be used as a less-efficient start codon for initiating translation of DPY-11. Our results also provide direct evidence that the body hypodermis and male tail of C. elegans have differential requirements of dpy-11 activity for their respective normal morphogenesis.

Alleles↗
PubMed2000

Molecular dynamics of the anticodon domain of yeast tRNA(Phe): codon-anticodon interaction.

We have studied the effect of codon-anticodon interaction on the structure and dynamics of transfer RNAs using molecular dynamics simulations over a nanosecond time scale. From our molecular dynamical investigations of the solvated anticodon domain of yeast tRNA(Phe) in the presence and absence of the codon trinucleotides UUC and UUU, we find that, although at a gross level the structures are quite similar for the free and the bound domains, there are small but distinct differences in certain parts of the molecule, notably near the Y37 base. Comparison of the dynamics in terms of interatomic or inter-residual distance fluctuation for the free and the bound domains showed regions of enhanced rigidity in the loop region in the presence of codons. Because fluorescence experiments suggested the existence of multiple conformers of the anticodon domain, which interconvert on a much larger time scale than our simulations, we probed the conformational space using five independent trajectories of 500 ps duration. A generalized ergodic measure analysis of the trajectories revealed that at least for this time scale, all the trajectories populated separate parts of the conformational space, indicating a need for even longer simulations or enhanced sampling of the conformational space to give an unequivocal answer to this question.

Anticodon↗
PubMed2002

Single synonymous codon substitution eliminates pausing during chloramphenicol acetyl transferase synthesis on Escherichia coli ribosomes in vitro.

The coding sequence for chloramphenicol acetyl transferase (CAT) contains several rare codons; three of them are ATA encoding isoleucine in positions 13, 84 and 119 of the amino acid sequence. Expression of CAT on Escherichia coli ribosomes in vitro results in mostly full-length product but also distinct smaller polypeptides from less than 3 kDa to over 20 kDa. As reported earlier, the smaller polypeptides are the predominant products, if translation is initiated with fluorophore-Met-tRNA(f). All this translational pausing is eliminated when the first ATA codon is mutated to ATC, a frequently used codon for isoleucine in E. coli. Addition of large amounts of E. coli tRNA to the coupled transcription/translation reaction does not reduce the number of pause-site peptides seen in the expression of wild-type CAT. Thus we hypothesize that the mRNA structure may be an important determinant for translational pausing.

Base Sequence↗
PubMed1990

Late onset adrenal hyperplasia: mutation at codon 282 of the functional 21-hydroxylase gene is not ubiquitous.

Ten patients affected with 21-hydroxylase (21-OH) deficient late-onset adrenal hyperplasia were studied to determine the prevalence of a mutation at codon 281 of the functional 21-OH gene (CYP21B) that results in a valine to leucine amino acid shift. This mutation has been reported in eight unrelated late-onset adrenal hyperplasia patients of Ashkenazi Jewish descent possessing the human leukocyte antigen-B14,DR1 haplotype. Normally, there are two 21-OH genes; a pseudogene (CYP21A) and a functional gene (CYP21B). The aberrant codon 281 sequence is normally present only in CYP21A. In all of our late-onset adrenal hyperplasia patients, hybridization of an oligonucleotide probe specific for this mutation was demonstrated to CYP21A but not to CYP21B. The mutation at codon 281 of CYP21B does not appear to be a ubiquitous genetic marker for 21-OH deficient late-onset adrenal hyperplasia, suggesting that this disorder may demonstrate the same molecular heterogeneity as congenital adrenal hyperplasia.

Adrenal Glands↗
PubMed1999

Codon usage in Plasmodium vivax nuclear genes.

Codon usage in Plasmodium vivax nuclear genes was analysed and compared with that in Plasmodium falciparum nuclear genes. Preferred codons were determined for P. vivax. Unlike P. falciparum, P. vivax genes are about 15% less A+T rich in the coding regions, with no obvious A+T bias at the third position of the codons. The amino-acid composition of P. vivax gene products is also different from that of P. falciparum. These results provide valuable information to facilitate gene cloning as well as expression and transfection studies for P. vivax.

Amino Acids↗
PubMed1986

Advantages to mutagenesis techniques generating populations containing the complete spectrum of single codon changes.

The limitations of current mutagenesis techniques are analyzed in terms of the number and kinds of codon changes they make and in terms of the population size needed to produce all single or multiple amino acid variants. It is shown how a technique that can alter a single codon of a gene, producing all possible variant codons without affecting the rest of the gene, has certain advantages, if it can be used at each place in the gene in one experiment. Such a technique has advantages when the goals are to understand: (1) how specific structural alterations in a mutant protein cause it to function in a different but specific way, (2) how to predict which amino acids in a protein contact or interact with each other, and (3) why a protein is more or less sensitive to mutational disruption, depending upon the specific mutation. This is because it would generate the maximum number of (1) mutant proteins with different functions, (2) intracistronic suppressor for any starting mutation, and (3) random amino acid substitutions at random places. Furthermore, such a technique could produce useful variants more quickly and on a smaller scale than either evolution or current methods.

Amino Acids↗
PubMed1999

Codon 249 of the human TP53 tumor suppressor gene is no hot spot for aflatoxin B1 in a heterologous background.

Mutations in the TP53 tumor suppressor gene are the most common alteration in cancer, and human primary liver cancers related to previous dietary exposure to the mycotoxin aflatoxin B1 (AFB1) exhibit a specific hot spot mutation at TP53 codon 249. We have asked whether the 249 hot spot is related to a particular susceptibility to AFB1 of this TP53 region or whether it is related to a phenotype of the 249S p53 mutant protein. This was addressed by constructing a metabolically competent variant of Saccharomyces cerevisiae strain yIG397 expressing human cytochrome P450 1A2 and P450-reductase and isolating AFB1-induced mutants that failed to express the genomic ADE2 reporter gene. Molecular analysis revealed that only 8/40 mutants had a mutation in the TP53 target gene, whereas 32/40 mutants were due to a recombination event eliminating the ADE2 reporter gene. None of 19 mutations identified in the eight mutant TP53 plasmids altered codon 249, thus this codon was no hot spot if the TP53 gene was in the heterologous background yeast. The genotoxic action of AFB1 was completely different from that of the alkylating agent ethyl-methane-sulfonate, where 28/30 induced mutations were linked to the TP53 target gene.

Aflatoxin B1↗
PubMed1996

Nonsense mutations inhibit RNA splicing in a cell-free system: recognition of mutant codon is independent of protein synthesis.

Mutations resulting in premature termination codons reduce the corresponding mRNA levels. We describe a cell-free system in which depletion of the mutant immunoglobulin kappa mRNA pool correlates with inefficient splicing and not with RNA decay. Splicing deficiency does not depend on the sequence surrounding the in-frame nonsense codon and can be partially corrected by mutating the methionine initiation codon. Despite the apparent link between translation and low mutant mRNA levels, inefficient splicing is not dependent on protein synthesis. Abnormal splicing of mutant immunoglobulin RNA is observed with B-cell but not with HeLa or T-cell extracts. A nonsense mutant beta-globin RNA is normally spliced by B-cell extract. We propose that the phenomenon exhibits tissue and gene specificity.

Animals↗
PubMed2003

Distribution of codon 129 genotype in human growth hormone-treated CJD patients in France and the UK.

Since homozygosity MM at codon 129 of the prion protein gene is a recognised risk factor in all forms of Creutzfeldt-Jakob disease (CJD), we studied the distribution of codon 129 polymorphism in patients in France and in the UK with CJD transmitted iatrogenically by human growth hormone. The overall frequencies of codon 129 genotypes in these patients differed from those in the population unaffected by CJD. An excess of VV homozygotes was noted among those with iatrogenic CJD compared with sporadic CJD cases. The proportion of MM genotype in UK patients was surprisingly low (4%) compared with that in French patients (62%). There is no evident explanation for this different distribution, which might be due to infection with different strains of prion in human growth hormone.

Codon↗
PubMed1998

p53 codon 72 polymorphism and risk of cervical cancer in UK.

BACKGROUND: A polymorphism at codon 72 of the human tumour-suppressor gene, p53, results in translation to either arginine or proline. A recent report suggested that the risk of human-papillomavirus-associated cervical cancer in white women is higher for those homozygous for the arginine allele than for those who are heterozygous. We examined a similar number of cervical cancers and a larger control group for their p53 codon 72 polymorphism status to see if we could confirm this result. METHODS: Three different groups of UK white women were studied: 96 who had volunteered to take part in a trial of ovarian-cancer screening; 150 attending for routine antenatal care in the Oxford region; and 50 women with cervical cancer. DNA from peripheral blood samples and from archival tissue samples was examined by PCR with allele-specific primers. FINDINGS: The proportions of individuals homozygous for the arginine allele, homozygous for the proline allele, and heterozygous for the two alleles were 59%, 4%, and 36% among women screened for ovarian cancer; 65%, 8%, and 27% among the antenatal-care group; and 54%, 6%, and 40% in women with cervical cancer. Chi2 analysis showed no significant differences in these proportions. INTERPRETATION: In the population studied, individuals homozygous for the arginine variant of codon 72 of the p53 gene were not at increased risk of cervical cancer.

Amino Acid Sequence↗
PubMed1996

High incidence of a T nucleotide at the second position of codon 97 in Vdelta2-(D)-Jdelta1 junctional sequences of human normal skin gamma delta T-cells.

In this investigation, we have demonstrated that in-frame polyclonal Vdelta2-(D)-Jdelta1 junctional sequences from human skin gammadelta T-cells contain a high incidence of T nucleotides at the second position of codon 97. Analysis of the deduced amino acid sequences based on in-frame Vdelta2-(D)-Jdelta1 junctional nucleotide sequences from normal skin gammadelta T-cells revealed a high incidence of the amino acids valine and leucine at position 97. These results are consistent with the studies in peripheral blood gammadelta T-cells, but not with previous findings in skin gammadelta T-cells, where random nucleotides were observed in the second position of codon 97 of Vdelta2-(D)-Jdelta1 junctional sequences and only a small minority of the deduced Vdelta2-(D)-Jdelta 1 amino acid sequences showed the amino acids valine and leucine at codon 97. Therefore, our findings indicate that the human skin gamma delta T-cells with a T-cell receptor consisting of a Vdelta2-(D)-Jdelta1-C delta1 chain are not a subset distinct from the subpopulation of human peripheral blood gammadelta T-cells expressing the same chain.

Codon↗