Codon Optimization

It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease.

The ongoing global threat posed by SARS-CoV-2 necessitates the rapid development of effective vaccines. This study employed a computational pipeline to design a multiepitope vaccine targeting the spike (S) glycoprotein of SARS-CoV-2. Cytotoxic T lymphocyte (CTL) epitopes were predicted using immunoinformatics tools and were screened based on their non-toxicity, immunogenicity, and antigenicity. High-affinity epitopes were sequentially linked via AAY linkers to construct a rationally designed vaccine candidate. The tertiary structure of the construct was modeled and evaluated for structural stability and desirable physicochemical properties. To assess immunogenic potential, molecular docking was performed with key immune receptors, including Toll-like receptor 3 (TLR3) and major histocompatibility complex class I (MHC-I), demonstrating strong and specific binding interactions. Furthermore, the vaccine gene was codon-optimized and in silico cloned into the pET-28b(+) expression vector, yielding a construct of 5476 base pairs. The collective in silico findings support the designed multi-epitope vaccine as a promising candidate capable of inducing robust and long-lasting cell-mediated immunity against SARS-CoV-2. Experimental validation is warranted to confirm these computational predictions.

The efficient production of functional proteins in heterologous hosts is one of the major bases of modern biotechnology. Unfortunately, many genes are difficult to express outside their original context. Due to their apparent "silent" nature, synonymous codon substitutions have long been thought to be trivial. In recent years, this dogma has been refuted by evidence that codon replacement can have a significant impact on gene expression levels and protein folding. In the past decade, considerable advances in the speed and cost of gene synthesis have facilitated the complete redesign of entire gene sequences, dramatically improving the likelihood of high protein expression. This technology significantly impacts the economic feasibility of microbial-based biotechnological processes by, for example, increasing the volumetric productivities of recombinant proteins or facilitating the redesign of novel biosynthetic routes for the production of metabolites. This review discusses the current applications of this technology, particularly those regarding the production of small molecules and industrially relevant recombinant enzymes. Suggestions for future research and potential uses are provided as well.

It is proposed that the X circular code, a set of 20 trinucleotides (codons) that has been identified in the protein-coding genes of most organisms from bacteria to eukaryotes, represents a new genetic signal that contributes to the regulation of gene expression. Circular codes have the important mathematical property of being error-correcting codes, and it has been hypothesized that the X circular code represents an ancestor of the standard genetic code, used in primordial systems to simultaneously decode amino acids and synchronize the reading frame. Here, using previously published experimental data, we demonstrate important correlations between the presence of X motifs in genes and more efficient gene expression. Our contributions suggest that X motifs continue to play a functional role in extant genomes and represent a new genetic signal, contributing to the maintenance of the correct reading frame and the optimization and regulation of gene expression.

Import of the precursor to 5-enolpyruvylshikimate-3phosphate synthase (pEPSPS) into chloroplasts is inhibited by the herbicide glyphosate. Inhibition of import is maximal at glyphosate concentrations of 2 10 itm and occurs only when pEPSPS is present as a ternary complex of enzyme-shikimate-3-phosphate-glyphosate. Glyphosate alone had no effect on the import of pEPSPS since it is not known to interact with the enzyme in the absence of shikimate-3-phosphate. Experiments with wild-type and glyphosate-resistant mutant forms of pEPSPS show that inhibition of import is directly proportional to the binding constants for glyphosate. Inhibition of import is thus a direct consequence of glyphosate binding to the enzymeshikimate-3-phosphate complex. The potential for non-specific effects of glyphosate on the chloroplast transport mechanism has been discounted by showing that import of another chloroplast-designated protein was unaffected by high concentrations of glyphosate and shikimate-3-phosphate. The mechanism of import inhibition by glyphosate is consistent with a precursor unfolding/refolding model.

DNA vaccine can be modified to increase protein production and modulate immune response. To enhance the efficiency of a P815 mastocytoma DNA vaccine, the P1A gene sequence was optimized by substituting specific codons with synonymous ones while modulating the number of CpG motifs. The P815A murine antigen production was increased with codonoptimized plasmids. The number of CpG motifs within the P1A gene sequence modulated the immunogenicity by inducing a local increase in the cytokines involved in innate immunity. After prophylactic immunization with the optimized vaccines, tumor growth was significantly delayed and mice survival was improved. Consistently, a more pronounced intratumoral recruitment of CD8 + T cells and a memory response were observed. Therapeutic vaccination was able to delay tumor growth when the codon-optimized DNA vaccine containing the highest number of CpG motifs was used. Our data demonstrate the therapeutic potential of optimized P1A vaccine against P815 mastocytoma, and they show the dual role played by codon optimization on both protein production and innate immune activation.

OPTIMIZER is an on-line application that optimizes the codon usage of a gene to increase its expression level. Three methods of optimization are available: the 'one amino acid-one codon' method, a guided random method based on a Monte Carlo algorithm, and a new method designed to maximize the optimization with the fewest changes in the query sequence. One of the main features of OPTIMIZER is that it makes it possible to optimize a DNA sequence using pre-computed codon usage tables from a predicted group of highly expressed genes from more than 150 prokaryotic species under strong translational selection. These groups of highly expressed genes have been predicted using a new iterative algorithm. In addition, users can use, as a reference set, a precomputed table containing the mean codon usage of ribosomal protein genes and, as a novelty, the tRNA gene-copy numbers. OPTIMIZER is accessible free of charge at http://genomes.urv.es/ OPTIMIZER.

A DNA fragment about 1.5 kb has been isolated from spleen of adult Chinese swine by RT-PCR. The DNA fragment encodes immunoglobulin IgG H chain gene. Sequencing analysis showed that the DNA fragment is 1 425 bp long, complete CDS. The C region of the gene has been classified as Subclass lg yj , and is the same as reported by Sun et al. , but V region of the present gene is 42 bp less by comparison. The gene has been ligated into expression vector PET-3b (NSEB) (-). A protein about 52 ku has been expressed in E. coli with an expression level of about 21 %. Keywords : swine, IgG H chnln gene, V region gene, doning , DNA sequencing, expression. SWINE is an important domestic animal in agriculture and even more promising to basic and clinical * The nucleotides sequence data reported in thin note have heen submitted to GenBank and assigned the accession numher ZHAOKAI AF062384. This note is a part of the firjt author's I'h. I). dissenation .

Cells control the efficiency of translation with various protein regulatory factors. Using computational approaches, both Tuller et al. and Cannarozzi et al. now find that translational efficiency is also hardwired into the transcript sequence. Tuller et al. show that messages often start with codons that correspond to rare transfer RNAs (tRNAs). This forms an early elongation "ramp" that fosters slow translation and may maximize the efficiency of protein production. Cannarozzi et al. find that once a codon for a particular amino acid is used, the subsequent codons for that amino acid are likely to be the same, despite the availability of multiple codons for each amino acid. This correlation is highly conserved across organisms and is shown to increase translation speed, suggesting that tRNAs are recycled and channeled by the ribosome.

Background: Breast cancer type 1 (BRCA1) gene also known as breast cancer type 1 susceptibility protein homologue plays important role in DNA double-strand break repair, homologous recombination, chromatin remodeling, cell cycle regulation and transcription regulation. Objective: Considering the importance of this protein, the present study was undertaken to analyze BRCA1 gene of different mammalian species by assessing the identity and similarity, phylogenetic relationship, physicochemical properties, predict the motifs, secondary and the tertiary structures. Materials and Methods: Seventeen nucleotide and protein sequences of BRCA1 gene of different mammalian species were retrieved from National Centre for Biotechnology Information (NCBI). Multiple sequence alignment was done using CLUSTAL W software, while identity and similarity was determined by constructing a pair wise comparison. Results: Results obtained at the end of the experiment showed that there was no percentage identity or similarity that was less than 70%. The phylogenetic relationship of BRCA1 gene of the mammalian species clustered into aquatic, herbivores, carnivores and omnivores, respectively. The highest time of divergence (95MYA) of the BRCA1 gene was observed between the BRCA1 gene of killer whale and human, while the least time of divergence (4.6MYA) was observed between the BRCA1 gene of cattle and American buffalo. Physicochemical properties of BRCA1 proteins in the five mammalian species (cattle, sheep, pig, American buffalo and human) were found to be unstable, hydrophilic and intracellular in nature. The following motifs were present at various positions in the BRCA1 gene of the five mammalian species; zinc finger RING, BRCT domain, N-myristoylation site, N-glycosylation, cAMP and cGMP dependent protein kinase phosphorylation site, tyrosine kinase phosphorylation, casein kinase 2 phosphorylation site, protein kinase C phosphorylation site and cell attachment sequence but leucine zipper pattern and microbodies c-terminal motifs were found in only human BRCA1 gene. The BRCA1 secondary structure contained the alpha helix, extended strand and random coil. Conclusion: Based on the results obtained, it can be deduced that BRCA1 gene has identical homologue, functional similarity and highly conserved in these mammalian species.

The present study evaluates immunogenicity and protection potency of a codon-optimized GRA1 DNA vaccine, wild type GRA1 DNA vaccine and an adjuvanted recombinant GRA1 protein vaccine candidate in BALB/c mice against lethal toxoplasmosis. Of the three GRA1 vaccines tested, the recombinant GRA1 protein vaccine results reveal significant increase in immune response and prolonged survival against acute toxoplasmosis compared to DNA vaccinations. Immune response and protection conferred by codon-optimized GRA1 DNA vaccine was slightly better than wild type GRA1 DNA vaccine.

Today, we are learning the language in which God created life. With this profound new knowledge, humankind is on the verge of gaining immense, new power to heal." "The revolution in molecular biology and biotechnology can be considered as a potential Revolution of Military Affairs (RMA)," states Colonel Michael Ainscough, MD, MPH (2). According to Andrew Krepinevich, who originally coined the term RMA, "technological advancement, incorporation of this new technology into military systems, military operational advancement, and organizational adaptation in a way that fundamentally alters the character and conduct of conflict" are the four components that make up an RMA. For instance, the Gulf War has been classified as the beginning of the space information warfare RMA. "From the technological advances in biotechnology, biowarfare with genetically engineered pathogens may constitute a future such RMA," says Ainscough (2).

The study describes the characterization of Interleukin-6 cDNA and essential promoter sequences of the Indian Water Buffalo (Bubalus bubalis) and expression of the recombinant IL-6 in Escherichia coli. Buffalo IL-6 shows very high nucleotide level identity of the cDNA (98.7%) and promoter (98%) sequences with the corresponding cattle sequences. All the major regulatory elements of IL-6 promoter like AP-1, Multiple Response Element, NF-IL6, ETS binding domain and NF-κB binding sites show absolute conservation. Basal level IL-6 mRNA is detected in organs like liver, lung and spleen. Concanavalin A stimulated splenocytes produced maximum IL-6 mRNA at 8 h poststimulation. Recombinant IL-6 production in JM109 (DE3) and BL21 (DE3) pLysS bacterial system is substantially enhanced by supplementation of rare codon tRNAs through co-transformation with a second plasmid. BL21 (DE3) pLysS strain is a more efficient producer of the IL-6 as it expressed twofold more protein than by JM109 (DE3) cells. The study shows high-level conservation of IL-6 regulatory and coding sequences between cattle and buffalo, and indicates the use of a common reagent for studying the effects of this cytokine in these species.

Weed infestation is one of the major biotic
stress factors that is responsible for yield loss in directseeded
rice (DSR). Herbicide-resistant rice has potential to
improve the efficiency of weed management under DSR.
Hence, the popular indica rice cultivar IR64, was genetically
modified using Agrobacterium-mediated transformation
with a codon-optimized CP4-EPSPS (5enolpyruvylshikimate-3-phosphate
synthase) gene, with
N-terminal chloroplast targeting peptide from Petunia
hybrida. Integration of the transgenes in the selected rice
plants was confirmed by Southern hybridization and
expression by Northern and herbicide tolerance assays.
Transgenic plants showed EPSPS enzyme activity even athigh concentrations of glyphosate, compared to untransformed
control plants. T
0
, T
1
and T
lines were tested by
herbicide bioassay and it was confirmed that the transgenic
rice could tolerate up to 1 % of commercial Roundup,
which is five times more in dose used to kill weeds under
field condition. All together, the transgenic rice plants
developed in the present study could be used efficiently to
overcome weed menace.