RESUMO
AIMS: How recoding of fnr, an anaerobic regulatory gene, affects pathogenicity related parameters of Salmonella Typhimurium (STM). METHODS AND RESULTS: The fnr gene was recoded by substituting all of it's codons with synonymous rare codons of STM. Recoding fnr gene severely reduced the ability of the recoded mutant to compete with wild strain under nutrient depletion condition. Mutants were also less motile than the wild strain and their biofilm forming ability was significantly decreased as compared to wild strain. The recoded strain showed significant reduced survival within murine macrophages (RAW264.7) and monocyte derived macrophage of poultry origin. The colonisation ability of recoded mutant in liver and spleen of mice on day 5 of post infection was significantly reduced. The recoded strain exhibited significant reduction in faecal shedding on day 1 and 5 after infection. CONCLUSIONS: Our study showed that recoding the anaerobic regulator fnr of STM significantly compromised its growth, decreased motility, biofilm forming ability and survival within macrophages. Further, the recoded fnr strain showed reduced colonisation ability and faecal shedding in mice. Thus, these findings highlight that recoding the global anaerobic regulator fnr of Salmonella Typhimurium attenuates its pathogenicity.
Assuntos
Genes Reguladores , Salmonella typhimurium , Anaerobiose , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Códon , Camundongos , VirulênciaRESUMO
OBJECTIVE: D-Glucosamine (GlcN) is an important amino sugar with various applications in medicine, food & beverages, nutritional supplements, and dairy products. This study aimed to produce GlcN from N-acetyl-D-glucosamine (GlcNAc) with an efficient deacetylase, and apply different strategies to enhance GlcN production. RESULTS: We screened a series of deacetylases that involved in the deacetylation of GlcNAc to form GlcN. A diacetylchitobiose deacetylase (TKDac) from Thermococcus kodakarensis exhibited high-efficient deacetylation activity for GlcNAc, yet mostly in the form of inclusion bodies. The soluble expression of TKDac was improved by a co-expressing molecular chaperone (groEL) and TKDac, and insertion of rare codon ATA encoding isoleucine. As such, the recombinant strain TKEL4 was constructed to express TKDac, and 48 g/L GlcN was achieved by TKDac-catalyzed deacetylation. To overcome the inhibition of byproduct (acetate), immobilized TKDac was carried out to produce GlcN from GlcNAc. The immobilized TKDac was conveniently re-used for several batches (above 8) with a 90% conversion rate. CONCLUSIONS: TKDac from T. kodakarensis was found to be an efficient deacetylase to produce GlcN. Co-expression of molecular chaperone and target protein, and insertion of rare codons were effective to improve the soluble expression of TKDac. The immobilized TKDac represents a promising method for future GlcN production.
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Acetilglucosamina , Glucosamina , Acetilglucosamina/metabolismo , Catálise , Glucosamina/metabolismoRESUMO
In Escherichia coli, the expression of heterologous genes for the production of recombinant proteins can be challenging due to the codon bias of different organisms. The rare codons AGG and AGA are among the rarest in E. coli. In this work, by using the human gene RioK2 as case study, we found that the presence of consecutive AGG-AGA led to a premature stop, which may be caused by an event of -1 frameshift. We found that translational problems caused by consecutive AGG-AGA are sequence dependent, in particular, in sequences that contain multiple rare AGG or AGA codons elsewhere. Translational problems can be alleviated by different strategies, including codon harmonization, codon optimization, or by substituting the consecutive AGG-AGA codons by more frequent arginine codons. Overall, our results furthered our understanding about the relationship between consecutive rare codons and translational problems. Such information will aid the design of DNA sequence for the production of recombinant proteins.
Assuntos
Códon , Escherichia coli/metabolismo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência de Arginina/genética , Proteínas Recombinantes/metabolismo , Escherichia coli/genética , Humanos , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Ribossomos/metabolismoRESUMO
Hepatitis C virus (HCV) infects liver cells and often causes chronic infection, also leading to liver cirrhosis and cancer. In the cytoplasm, the viral structural and non-structural (NS) proteins are directly translated from the plus strand HCV RNA genome. The viral proteins NS3 to NS5B proteins constitute the replication complex that is required for RNA genome replication via a minus strand antigenome. The most C-terminal protein in the genome is the NS5B replicase, which needs to initiate antigenome RNA synthesis at the very 3'-end of the plus strand. Using ribosome profiling of cells replicating full-length infectious HCV genomes, we uncovered that ribosomes accumulate at the HCV stop codon and about 30 nucleotides upstream of it. This pausing is due to the presence of conserved rare, inefficient Wobble codons upstream of the termination site. Synonymous substitution of these inefficient codons to efficient codons has negative consequences for viral RNA replication but not for viral protein synthesis. This pausing may allow the enzymatically active replicase core to find its genuine RNA template in cis, while the protein is still held in place by being stuck with its C-terminus in the exit tunnel of the paused ribosome.
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Códon , Genoma Viral , Hepacivirus/fisiologia , Fases de Leitura Aberta , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/biossíntese , Ribossomos/metabolismo , Replicação Viral/fisiologia , Linhagem Celular Tumoral , Humanos , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Ribossomos/genéticaRESUMO
P-element-induced wimpy-like (Piwil) proteins restrict the replication of mobile genetic elements in the germ line. They are also expressed in many transformed cell lines. In this study, we discovered that the human Piwil 2 (Hili) protein can also inhibit HIV replication, especially in activated CD4+ T cells that are the preferred target cells for this virus in the infected host. Although resting cells did not express Hili, its expression was rapidly induced following T cell activation. In these cells and transformed cell lines, depletion of Hili increased levels of viral proteins and new viral particles. Further studies revealed that Hili binds to tRNA. Some of the tRNAs represent rare tRNA species, whose codons are overrepresented in the viral genome. Targeting tRNAArg(UCU) with an antisense oligonucleotide replicated effects of Hili and also inhibited HIV replication. Finally, Hili also inhibited the retrotransposition of the endogenous intracysternal A particle (IAP) by a similar mechanism. Thus, Hili joins a list of host proteins that inhibit the replication of HIV and other mobile genetic elements.IMPORTANCE Piwil proteins inhibit the movement of mobile genetic elements in the germ line. In their absence, sperm does not form and male mice are sterile. This inhibition is thought to occur via small Piwi-interacting RNAs (piRNAs). However, in some species and in human somatic cells, Piwil proteins bind primarily to tRNA. In this report, we demonstrate that human Piwil proteins, especially Hili, not only bind to select tRNA species, including rare tRNAs, but also inhibit HIV replication. Importantly, T cell activation induces the expression of Hili in CD4+ T cells. Since Hili also inhibited the movement of an endogenous retrovirus (IAP), our finding shed new light on this intracellular resistance to exogenous and endogenous retroviruses as well as other mobile genetic elements.
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Proteínas Argonautas/metabolismo , HIV-1/fisiologia , Ativação Linfocitária , Replicação Viral , Proteínas Argonautas/deficiência , Proteínas Argonautas/genética , Proteínas Argonautas/imunologia , Linhagem Celular , Retrovirus Endógenos/metabolismo , Células HEK293 , HIV-1/genética , Células HeLa , Humanos , Oligonucleotídeos Antissenso/genética , Ligação Proteica , RNA Interferente Pequeno/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência de Arginina/genética , RNA de Transferência de Arginina/metabolismo , Linfócitos T/virologiaRESUMO
Codon bias at the 5' terminal of coding sequence (CDS) is known to be distinct from the rest of the CDS. A number of events occur in this short region to regulate early translation elongation and co-translational translocation. In the genes encoding secretory proteins, there is a special signal sequence which has a higher occurrence of rare codons. In this study, we analyzed codon bias of secretory genes in several eukaryotes. The results showed that secretory genes in the species except mammals had a higher occurrence of rare codons in the 5' terminal of CDS, and the bias was greater than the same region of non-secretory genes. GO analysis revealed that secretory genes containing rare codon clusters in different regions were responsible for various roles in gene functions. Moreover, codon bias in the region encoding the hydrophobic region of protein is similar in secretory and non-secretory genes, indicating that codon bias in secretory genes was partly influenced by amino acid bias. Rare codon clusters are found more frequently in specific regions, and continuous rare codons are not favoured probably because they will increase the probability of ribosome collision and drop-off. Based on ribosome profiling data, there is no significant difference in the average translation efficiencies between rare and optimal codons. Higher ribosomal density in the 5' terminal may result from ribosome pausing which could be involved in different translation events. These findings collectively provided rich information on codon bias in secretory genes, which may shed light on the co-effect of codon bias, mRNA structure and tRNA abundance in translational regulations.
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Códon/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Biologia Computacional/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fases de Leitura Aberta , Plantas/genética , RNA Mensageiro/química , Ribossomos/genética , Saccharomyces cerevisiae/genéticaRESUMO
Rare codon in a heterologous gene may cause premature termination of protein synthesis, misincorporation of amino acids, and/or slow translation of mRNA, decreasing the heterologous protein expression. However, its hypothetical function pertaining to functional protein folding has been barely reported. Here, we investigated the effects of selective introduction of synonymous rare codons (SRCs) to two codon-optimized (i.e., rare codon-free) genes sucrose phosphorylase (SP) gene from Thermoanaerobacterium thermosaccharolyticum and amidohydrolase gene from Streptomyces caatingaensis on their expression levels in Escherichia coli BL21(DE3). We investigated the introduction of a single SRC to the coding regions of alpha-helix, beta-strand, or linker in the first half of rare codon-free sp and ah gene. The introduction of a single SRC in the beginning of the coding regions of beta-strand greatly enhanced their soluble expression levels as compared to the other regions. Also, we applied directed evolution to test multi-SRC-containing sp gene mutants for enhanced soluble SP expression levels. To easily identify the soluble SP expression level of colonies growing on Petri dishes, mCherry fluorescent protein was used as a SP-folding reporter when it was fused to the 3' end of the sp gene mutant libraries. After three rounds of screening, the best sp gene mutant containing nine SRCs exhibited an approximately six-fold enhancement in soluble protein expression level as compared to the wild-type and rare codon-free sp control. This study suggests that the selective introduction of SRCs can attenuate translation at specific points and such discontinuous attenuation can temporally separate the translation of segments of the peptide chains and actively coordinates their co-translational folding, resulting in enhanced functional protein expression. Biotechnol. Bioeng. 2017;114: 1054-1064. © 2016 Wiley Periodicals, Inc.
Assuntos
Códon/genética , Evolução Molecular Direcionada/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Glucosiltransferases/genética , Mutação Silenciosa/genética , Clonagem Molecular , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Modelos Moleculares , Biossíntese de Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Thermoanaerobacterium/enzimologia , Thermoanaerobacterium/genéticaRESUMO
In well-established heterologous hosts, such as Escherichia coli, recombinant proteins are usually intracellular and frequently found as inclusion bodies-especially proteins possessing high rare codon content. In this study, successful secretory expression of three hydrolases, in a constructed inducible or constitutive system, was achieved by fusion with a novel signal peptide (Kp-SP) from an actinomycete. The signal peptide efficiently enabled extracellular protein secretion and also contributed to the active expression of the intracellular recombinant proteins. The thermophilic α-amylase gene of Bacillus licheniformis was fused with Kp-SP. Both recombinants, carrying inducible and constitutive plasmids, showed remarkable increases in extracellular and intracellular amylolytic activity. Amylase activity was observed to be > 10-fold in recombinant cultures with the constitutive plasmid, pBSPPc, compared to that in recombinants lacking Kp-SP. Further, the signal peptide enabled efficient secretion of a thermophilic cellulase into the culture medium, as demonstrated by larger halo zones and increased enzymatic activities detected in both constructs from different plasmids. For heterologous proteins with a high proportion of rare codons, it is difficult to obtain high expression in E. coli owing to the codon bias. Here, the fusion of an archaeal homologue of the amylase encoding gene, FSA, with Kp-SP resulted in > 5-fold higher extracellular activity. The successful extracellular expression of the amylase indicated that the signal peptide also contributed significantly to its active expression and signified the potential value of this novel and versatile signal peptide in recombinant protein production.
Assuntos
Actinobacteria/genética , Bacillus subtilis/genética , Proteínas de Bactérias , Sistemas de Secreção Bacterianos , Escherichia coli , Sinais Direcionadores de Proteínas , alfa-Amilases , Bacillus subtilis/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Sistemas de Secreção Bacterianos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , alfa-Amilases/biossíntese , alfa-Amilases/genéticaRESUMO
Presence of the rare codons resulted from the difference in codon usages among organisms is considered as an obstacle to heterologous gene expression. This is especially important for the expression of the genes with eukaryotic origin in Escherichia coli. The N-terminus of human granulocyte colony stimulating factor (hG-CSF) contains amino acids whose coding sequences belong to the rare codons in E. coli. In this study, the effect of rare codons on hG-CSF expression level was evaluated through introducing silent mutations in the 5'-end of the coding sequence. E. coli BL21 (DE3) was used as an expression host. The constructs with the rare codons at the positions following the ATG initiation site of hG-CSF elevated the expression level up to 53-56% of the total cell proteins. This effect may be explained either by the rare codons effects on the early elongation region to reduce ribosome traffic jams in the rest of transcript or by their impacts on reduction of GC content at the beginning region. Mfold RNA server and prediction of the 5' mRNA secondary structure showed the less stable mRNA secondary structure is, the more hG-CSF expression level would be. However, the minimum free energy of the secondary structure individually, could not indicate this correlation between all constructs. This finding seems empirically important in designing the synthetic genes for production of the recombinant protein in E. coli.
Assuntos
Códon de Iniciação/genética , Códon/genética , Escherichia coli/genética , Fator Estimulador de Colônias de Granulócitos/genética , Proteínas Recombinantes/genética , Fator Estimulador de Colônias de Granulócitos/química , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The Gram-positive bacterium Bacillus megaterium was systematically developed for the plasmid-based production of recombinant proteins at the gram-per-liter scale. The amount of protein produced per cell was found strongly correlated to the codon usage of the heterologous gene of interest in comparison to the codon usage of B. megaterium. For analyzing the influence of rare codons on the translational efficiency and protein production in B. megaterium, a test system using the gene for the green fluorescent protein (GFP) as reporter was established. For this purpose, four consecutive identical codons were introduced into the 5' end of gfp and the resulting variations in GFP formation were quantified. Introduction of the rare codons GCC, CGG, and ACC for alanine, arginine, and threonine reduced GFP production 2.1-, 3.3-, and 1.7-fold in comparison to the favored codons GCU, CGU, and ACA, respectively. Coexpression of the corresponding rare codon tRNA (rctRNA) genes improved GFP production 4.2-, 2.7-, and 1.7-fold, respectively. The system was applied to the production of a formate dehydrogenase (FDH) from Mycobacterium vaccae and an extracellular hydrolase (TFH) from Thermobifida fusca. Coexpression of one to three different rctRNA genes resulted in an up to 18-fold increased protein production. Interestingly, rctRNA gene coexpression also elevated the production of M. vaccae FDH and T. fusca TFH from codon optimized genes, indicating a general positive effect by rctRNA gene overexpression on the protein production in B. megaterium. Thus, the basis for a B. megaterium enhanced production strain coexpressing rctRNA genes was laid.
Assuntos
Bacillus megaterium/metabolismo , Códon , Engenharia Metabólica/métodos , Biossíntese de Proteínas , Engenharia de Proteínas/métodos , RNA de Transferência/metabolismo , Proteínas Recombinantes/biossíntese , Actinobacteria/enzimologia , Actinobacteria/genética , Bacillus megaterium/genética , Formiato Desidrogenases/genética , Formiato Desidrogenases/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Hidrolases/genética , Hidrolases/metabolismo , Mycobacterium/enzimologia , Mycobacterium/genética , RNA de Transferência/genética , Proteínas Recombinantes/genéticaRESUMO
BACKGROUND: Herpes simplex virus type 1 (HSV-1) is associated with Alzheimer's disease, which goes into a cycle of latency and reactivation. The present study was envisaged to understand the reasons for latency and specific molecular patterns present in the HSV-1. OBJECTIVE: The objective is the molecular dissection of Herpes simplex virus type 1 to elucidate molecular mechanisms behind latency and compare its codon usage patterns with genes modulated during Alzheimer's disease as a part of host-pathogen interaction. METHODS: In the present study, we tried to investigate the potential reasons for the latency of HSV-1 virus bioinformatically by determining the CpG patterns. Also, we investigated the codon usage pattern, the presence of rare codons, codon context, and protein properties. RESULTS: The top 222 codon pairs graded based on their frequency in the HSV-1 genome revealed that with only one exception (CUG-UUU), all other codon pairs have codons ending with G/C. Considering it an extension of host-pathogen interaction, we compared HSV-1 codon usage with that of codon usage of genes modulated during Alzheimer's disease, and we found that CGT and TTT are only two codons that exhibited similar codon usage patterns and other codons showed statistically highly significant different codon preferences. Dinucleotide CpG tends to mutate to TpG, suggesting the presence of mutational forces and the imperative role of CpG methylation in HSV-1 latency. CONCLUSIONS: Upon comparison of codon usage between HSV-1 and Alzheimer's disease genes, no similarities in codon usage were found as a part of host-pathogen interaction. CpG methylation plays an imperative role in latency HSV-1.
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Doença de Alzheimer , Herpes Simples , Herpesvirus Humano 1 , Humanos , Herpesvirus Humano 1/genética , Uso do Códon , Doença de Alzheimer/genética , Interações Hospedeiro-Patógeno/genética , Herpes Simples/metabolismoRESUMO
OBJECTIVE: Rare codons were previously shown to be enriched at the beginning of the dengue virus (DENV) open reading frame. However, the role of rare codons in regulating translation efficiency and replication of DENV remains unclear. The present study aims to clarify the significance of rare codon usage at the beginning of DENV transcripts using the codon adaptation index (CAI). METHODOLOGY: CAIs of the whole starting regions of DENV transcripts as well as 18-codon sliding windows of the regions were analyzed. RESULTS: One of the intriguing findings is that those rare codons do not typically result in uniformly low CAI in the starting region with rare codons. However, it shows a notable local drop in CAI around the 50th codon in all dengue serotypes. This suggests that there may be a translational checkpoint at this site and that the rare codon usage upstream to this checkpoint may not be related to translational control.
Assuntos
Uso do Códon , Vírus da Dengue , Vírus da Dengue/genética , Fases de Leitura Aberta/genética , Códon/genéticaRESUMO
Background: L-lysine is widely used in the feed, food, and pharmaceutical industries, and screening for high L-lysine-producing strains has become a key goal for the industry. Methods: We constructed the rare L-lysine codon AAA by corresponding tRNA promoter replacement in C. glutamicum. Additionally, a screening marker related to the intracellular L-lysine content was constructed by converting all L-lysine codons of enhanced green fluorescent protein (EGFP) into the artificial rare codon AAA. The artificial EGFP was then ligated into pEC-XK99E and transformed into competent Corynebacterium glutamicum 23604 cells with the rare L-lysine codon. After atmospheric and room-temperature plasma mutation and induction culture, 55 mutants (0.01% of total cells) with stronger fluorescence were sorted using flow cytometry, and further screened by fermentation in a 96-deep-well plate and 500 mL shaker. Results: The fermentation results showed that the L-lysine production was increased by up to 9.7% in the mutant strains with higher fluorescence intensities, and that the highest screening positive rate was 69%, compared with that in the wild-type strain. Conclusion: The application of artificially constructed rare codons in this study represents an efficient, accurate, and simple method for screening other amino acid-producing microorganisms.
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The SARS-CoV-2 delta variant (B.1.617.2) appeared for the first time in December 2020 and later spread worldwide. Currently available vaccines are not so efficacious in curbing the viral pathogenesis of the delta strain of COVID; therefore, the development of a safe and effective vaccine is required. In the present study, we envisaged molecular patterns in the structural genes' spike, nucleoprotein, membrane, and envelope of the SARS-CoV-2 delta variant. The study was based on determining compositional features, dinucleotide odds ratio, synonymous codon usage, positive and negative codon contexts, rare codons, and insight into relatedness between the human host isoacceptor tRNA and preferred codons from the structural genes. We found specific patterns, including a significant abundance of T nucleotide over all other three nucleotides. The underrepresentation of GpA, GpG, CpC, and CpG dinucleotides and the overrepresentation of TpT, ApA, CpT, and TpG were observed. A preference towards ACT- (Thr), AAT- (Asn), TTT- (Phe), and TTG- (Leu) initiated codons and aversion towards CGG (Arg), CCG (Pro), and CAC (His) was present in the structural genes of the delta strain. The interaction between the host tRNA pool and preferred codons of the envisaged structural genes revealed that the virus preferred the codons for those suboptimal numbers of isoacceptor tRNA were present. We see this as a strategy adapted by the virus to keep the translation rate low to facilitate the correct folding of viral proteins. The information generated in the study helps design the attenuated vaccine candidate against the SARS-CoV-2 delta variant using a synthetic biology approach. Three strategies were tested: changing TpT to TpA, introducing rare codons, and disrupting favored codons. It found that disrupting favored codons is a better approach to reducing virus fitness and attenuating SARS-CoV-2 delta strain using structural genes.
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Although synonymous mutations have long been thought to lack striking results, a growing body of research shows these mutations have highly variable effects. In this study, the impact of synonymous mutations in the development of thermostable luciferase was investigated using a combination of experimental and theoretical approaches. Using bioinformatics analysis, the codon usage features in the Lampyridae family's luciferases were studied and four synonymous mutations of Arg in luciferase were created. An exciting result was that the analysis of kinetic parameters showed a slight increase in the thermal stability of the mutant luciferase. AutoDock Vina, %MinMax algorithm, and UNAFold Server were used to perform molecular docking, folding rate, and RNA folding, respectively. Here, it was assumed that in the region (Arg337) with a moderate propensity for coil, synonymous mutation altered the rate of translation, which in turn may lead to a slight change in the structure of the enzyme. According to the molecular dynamics simulation data, local minor global flexibility is observed in the context of the protein conformation. A plausible explanation is that this flexibility may strengthen hydrophobic interactions due to its sensitivity to a molecular collision. Accordingly, thermostability originated mainly from hydrophobic interaction.
Assuntos
Simulação de Dinâmica Molecular , Mutação Silenciosa , Simulação de Acoplamento Molecular , Luciferases de Vaga-Lume/metabolismo , Dobramento de RNARESUMO
Aquacultural shrimps suffer economic lost due to the white spot syndrome virus (WSSV) that is the most notorious virus for its fatality and contagion, leading to a 100% death rate on infected shrimps within 7 days. However, the infection of mechanism remains a mystery and crucial problem. To elucidate the pathogenesis of WSSV, a high abundance of protein is required to identify and characterize its functions. Therefore, the optimal WSSV355 overexpression was explored in engineered Escherichia coli strains, in particular C43(DE3) as a toxic tolerance strain remedied 40% of cell growth from BL21(DE3). Meanwhile, a trace amount of WSSV355 was observed in both strains. To optimize the codon of WSSV355 using codon adaption index (CAI), an overexpression was observed with 1.32 mg/mL in C43(DE3), while the biomass was decreased by 35%. Subsequently, the co-expression with pRARE boosted the target protein up to 1.93 mg/mL. Finally, by scaling up production of WSSV355 in the fermenter with sufficient oxygen supplied, the biomass and total and soluble protein were enhanced 67.6%, 44.9%, and 7.8% compared with that in flask condition. Herein, the current approach provides efficacious solutions to produce toxic proteins via codon usage, strain selection, and processing optimization by alleviating the burden and boosting protein production in E. coli.
Assuntos
Escherichia coli , Vírus da Síndrome da Mancha Branca 1 , Escherichia coli/genética , Escherichia coli/metabolismo , Vírus da Síndrome da Mancha Branca 1/genética , Proteínas de Membrana/genética , Códon/genética , Códon/metabolismoRESUMO
In almost all eubacteria, the AUA codon is translated by tRNAIle2 bearing lysidine at the wobble position. Lysidine is introduced by tRNAIle lysidine synthetase (TilS) via post-transcriptional modification of the cytidine of tRNAIle2 (CAU). Lactobacillus casei and Lactobacillus plantarum have tilS homologues and tRNAIle2 (CAU) genes. In addition, L. casei also has another tRNAIle2 gene with an UAU anticodon. L. plantarum has a tRNAIle (UAU)-like RNA. Here, we demonstrate that L. casei tRNAIle2 (UAU) is charged with isoleucine by L. casei isoleucyl-tRNA synthetase (IleRS) but not by L. plantarum IleRS, even though the amino acid identity of these two enzymes is over 60%. It has been reported that, in Mycoplasma mobile, which has its tRNAIle2 (UAU) but no tilS homologue, an Arg residue at position 865 of the IleRS is required for recognition of the UAU anticodon. This position is occupied by an Arg also in the IleRSs from both of the Lactobacillus species. Thus, other residues in L. casei, IleRS should also contribute to the recognition of tRNAIle2 (UAU). We found that a chimeric L. casei IleRS in which the N-terminal domain was replaced by the corresponding region of L. plantatarum IleRS has very low aminoacylation activity towards both tRNAIle2 (UAU) and tRNAIle1 (GAU). The A18G mutant had barely detectable aminoacylation activity towards either of the tRNAsIle . However, a double point mutant of A18G and G19N aminoacylated tRNAIle1 (GAU), but not tRNAIle2 (UAU). Our results suggest that, for L. casei IleRS, Ala18 and Gly19 also play a critical role in recognition of tRNAIle2 (UAU).
Assuntos
Anticódon , Lactobacillales , Anticódon/genética , Isoleucina/genética , Isoleucina-tRNA Ligase/química , Lactobacillales/genética , Lactobacillales/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência de Isoleucina/química , RNA de Transferência de Isoleucina/genética , RNA de Transferência de Isoleucina/metabolismoRESUMO
BACKGROUND: Cholestatic disorders are divided in the extra and intra-hepatic that created due to the severe liver diseases. ABCB11 encodes the bile salt export pump and this gene is mutated in several forms of intrahepatic cholestasis. So far, some molecular features of this gene was studies. OBJECTIVE: Using a developed web server, we identified high number of rare codons in this gene, and four cases were related to BSEP-deficient patients which can be used for drug design. MATERIAL AND METHODS: By in-silico modelling of ABCB11, some of rare codons in different locations of ATP8b1 gene were identified and evaluated. Using several web servers a number of mutations that converted non-rare codons to rare codon in these patients were identified. RESULTS: Some of these rare Codons were located at special positions by mutation of which, the new side chains do not seem suitable for protein structure and function. Furthermore, this mutation changed the protein folding rate that may have a critical role in proper folding. Thus, primary change of these codons contributes to BSEP deficiency. CONCLUSION: This work is a comprehensive analysis of rare codons of ABCB11 and assessment of a number of these rare codon in protein levels. Rare codons evaluation can enhance our understanding of ABCB11 structural protein of ABCB11, and help us to develop mutation-specific therapies in design of new drugs.
RESUMO
BACKGROUND: Progressive familial intrahepatic cholestases (PFIC) are a spectrum of autosomal progressive liver diseases developing to end-stage liver disease. ATP8B1 deficiency caused by mutations in ATP8B1 gene encoding a P-type ATPase leads to PFIC1. The gene for PFIC1 has been mapped on a 19-cM region of 18q21-q22, and a gene defect in ATP8B1 can cause deregulations in bile salt transporters through decreased expression and/or activity of FXR. Point mutations are the most common, with the majority being missense or nonsense mutations. In addition, approximately 15% of disease-causing ATP8B1 mutations are annotated as splicing disrupting alteration given that they are located at exon-intron borders. OBJECTIVE: Here, we describe the hidden layer of computational biology information of rare codons in ATP8B1, which can help us for drug design. METHODS: Some rare codons in different locations of ATP8b1 gene were identified using several web servers and by in-silico modelling of ATP8b1 in Phyre2 and I-TASSER server, some rare codons were evaluated. RESULTS: Some of these rare codons were located at special positions which seem to have a critical role in proper folding of ATP8b1 protein. Structural analysis showed that some of rare codons are related to mutations in ATP8B1 that are responsible for PFIC1 disease, which may have a critical role in ensuring the correct folding. CONCLUSION: Investigation of such hidden information can enhance our understanding of ATP8b1 folding. Moreover, studies of these rare codons help us to clarify their role in rational design of new and effective drugs.
RESUMO
Mitoviruses replicate in mitochondria of their host fungi. They have small RNA genomes that encompass a single ORF encoding the viral RdRp. Since UGA codons encode Trp in fungal mitochondria, the RdRp ORF of a typical mitovirus includes multiple UGA codons. In some mitoviruses, however, the ORF has no such codons, suggesting that these particular viruses may be under selective pressure to exclude them. In this report, new evidence is presented that host fungi whose mitoviruses have no or few UGA codons are distinctive in also having no or few UGA codons in their core mitochondrial genes. Thus, the relative exclusion of such codons in a subset of mitoviruses appears to reflect most fundamentally that UGA(Trp) is a rare mitochondrial codon in their particular hosts. The fact that UGA(Trp) is a rare mitochondrial codon in many fungi appears not to have been widely discussed to date.