Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli.

In a lacZ expression vector (pMC1403Plac), all 64 codons were introduced immediately 3' from the AUG initiation codon. The expression of the second codon variants was measured by immunoprecipitation of the plasmid-coded fusion proteins. A 15-fold difference in expression was found among the codon variants. No distinct correlation could be made with the level of tRNA corresponding to the codons and large differences were observed between synonymous codons that use the same tRNA. Therefore the effect of the second codon is likely to be due to the influence of its composing nucleotides, presumably on the structure of the ribosomal binding site. An analysis of the known sequences of a large number of Escherichia coli genes shows that the use of codons in the second position deviates strongly from the overall codon usage in E. coli. It is proposed that codon selection at the second position is not based on requirements of the gene product (a protein) but is determined by factors governing gene regulation at the initiation step of translation.     

基因表达水平与同义密码子使用关系的初步研究

提出一个预测基因表达水平和同义密码子使用的自洽信息聚类方法。将同义密码子分成最适密码子、非最适密码子和稀有密码子,认为三者的使用频率是调控基因表达水平的主要因素。基于这一观点,对Ｅｃｏｌｉ和Ｙｅａｓｔ两类生物的基因表达水平和密码子的使用,用自洽信息聚类方法进行了预测。发现高低表达基因明显分开,基因表达水平被分为四级;甚高表达基因（ＶＨ）、高表达基因（Ｈ）、较低表达基因（ＬＭ）和低表达基因（ＬＬ）;     

Optimization of codon usage of the envelope protein E2 gene from various genotypes of hepatitis C virus to predict the expression level in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Hepatitis C virus infection (HCV) alarmingly increases worldwide; it causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, so there is urgent need of developing effective and sufficient quantity of vaccine. HCV envelope protein E2 is the main target for developing as a vaccine candidate. Presently recombinant proteins can successfully be used as a vaccine for many diseases. This concern, it is challenging to produce sufficient quantities of many recombinant proteins from their expression hosts. One of the main factors affecting the success of expression of foreign genes in heterologous hosts is the divergence of codon usage of the target gene from that used in the expression system. In this study, we optimized the various genotypes of HCV envelope protein E2 gene according to the codon usage of Pichia pastoris and predicted the expression level. Synonymous codon usage of E2 adapted to that used by P. pastoris was estimated using the relative synonymous codon usage value (RSCU), codon adaptation index (CAI) and effective number of codon (ENC). The CAI of optimized HCV E2 sequences was enhanced from 0.638 to 0.833 and %GC was decreased from 56.05 to 44.05; this was significantly (p < 0.01) different from the native sequences. Codon with RSCU value less than one was replaced with most preferred synonymous codons. The ENC values of optimized HCV E2 sequences varied from 47.00 to 47.50, with a mean value of 47.15 and an SD of 0.14. Our study suggested that, from the measured values of predicted expression level, the codon optimized HCV E2 protein could be produced in sufficient quantity in the expression host; knowledge of the codon usage patterns of E2 of various genotypes facilitate the production of a promising unique vaccine candidate for HCV.     

Enhanced expression of a recombinant malaria candidate vaccine in Escherichia coli by codon optimization

This study was conducted to compare the expression of three constructs of a multistage candidate vaccine (FALVAC-1) against Plasmodium falciparum in an Escherichia coli system: a synthetic gene with P. falciparum codons, a synthetic gene with optimized E. coli codons, and a synthetic gene with P. falciparum codons co-transformed with a RIG plasmid, which encodes three tRNAs (AG(A/G), ATA, GGA) that recognize rare E. coli codons. The expression of the protein increased at least threefold with codon optimization. The presence of the RIG plasmid in the co-transforming cells did not significantly increase the expression level of the gene with P. falciparum codons. The growth of cells transformed by the construct with P. falciparum codons was significantly slower than that of cells transformed by the construct with optimized E. coli codons after induction of protein expression with IPTG. The cells containing the non-codon optimized gene co-expressed with RIG plasmid had the slowest growth at all time points in culture. Thus, codon optimization significantly increases the yield of P. falciparum candidate vaccines in the E. coli expression system.     

Improvement of human interferon HUIFNalpha2 and HCV core protein expression levels in Escherichia coli but not of HUIFNalpha8 by using the tRNA(AGA/AGG)

High-level expression from one particular heterologous gene in Escherichia coli generally requires the optimization of codon usage. Genes encoding for Hepatitis C virus core protein (HCcAg), human interferon alpha2 and 8 subtypes (HUIFNalpha2 and HUIFNalpha8) show a high content of AGA/AGG codons. These are encoded by the product of the dnaY gene in E. coli. The proteins used in this work have a high therapeutic value and were used as models for studying the effects of these rare codons on the efficiency of heterologous gene expression in E. coli. Expression plasmids were constructed to express any of these proteins and the dnaY gene product simultaneously in E. coli. After dnaY gene expression, HCcAg, and HUIFNalpha2 expression levels increased 5 and 3 times, respectively. However, HUIFNalpha8 expression was barely detected either supplying or not the additional dnaY gene product. These results suggest that the high frequency of AGA/AGG codons present in the HCcAg and HUIFNalpha2 genes could be one of the factors limiting its expression in E. coli. Nevertheless, for HUIFNalpha8 it seems that other factors prevail upon the lack of dnaY product. Data presented here for HCcAg and HUIFNalpha2 expressions proved the value of this approach to obtain therapeutic proteins in E. coli.     

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.     

枯草芽孢杆菌同义密码子使用偏性对蛋白质折叠速率的影响

目前,有关同义密码子使用偏性对蛋白质折叠的影响研究中,样本蛋白均来源于不同的物种。考虑到同义密码子使用偏性的物种差异性,选取枯草杆菌的核蛋白为研究对象。首先,将每条核蛋白按二级结构截取为α螺旋片段、β折叠片段和无规卷曲（α-β混合）片段,并计算其蛋白质折叠速率。然后,整理每个片段相应的核酸序列信息,计算其同义密码子使用度。在此基础上,分析枯草芽孢杆菌核蛋白的同义密码子使用偏性与蛋白质折叠速率的相关性。发现对于不同二级结构的肽链片段,都有部分密码子的使用偏性与其对应的肽链折叠速率显著相关。进一步分析发现,与肽链片段折叠速率显著相关的密码子绝大部分为枯草杆菌全序列或核蛋白序列的每一组同义密码子中使用度最高的密码子。结果表明,在蛋白质的折叠过程中,枯草芽孢杆菌的同义密码子使用偏性起着重要作用。     

Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E. coli genome

High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms.     

Codon usage and gene expression.

The hypothesis that codon usage regulates gene expression at the level of translation is tested. Codon usage of Escherichia coli and phage lambda is compared by correspondence analysis, and the basis of this hypothesis is examined by connecting codon and tRNA distributions to polypeptide elongation kinetics. Both approaches indicate that if codon usage was random tRNA limitation would only affect the rarest tRNA species. General discrimination against their cognate codons indicates that polypeptide elongation rates are maintained constant. Thus, differences in expression of E. coli genes are not a consequence of their variable codon usage. The preference of codons recognized by the most abundant tRNAs in E. coli genes encoding abundant proteins is explained by a constraint on the cost of proof-reading.     

Codon usage in Entamoeba histolytica.

The codon usage of 10 E. histolytica genes comprising 4455 codons was analysed. The codon usage revealed an extremely biased use of synonymous codons with a preference for NNU (44%) and NNA (41.4%) codons. Codons CGG (arg), AGG (arg) and CCG (pro) were absent in the E. histolytica genes examined. The codon usage of E. histolytica resembled that of Plasmodium falciparum.     

The Effect of Multiple Evolutionary Selections on Synonymous Codon Usage of Genes in the Mycoplasma bovis Genome

Mycoplasma bovis is a major pathogen causing arthritis, respiratory disease and mastitis in cattle. A better understanding of its genetic features and evolution might represent evidences of surviving host environments. In this study, multiple factors influencing synonymous codon usage patterns in M. bovis (three strains’ genomes) were analyzed. The overall nucleotide content of genes in the M. bovis genome is AT-rich. Although the G and C contents at the third codon position of genes in the leading strand differ from those in the lagging strand (p<0.05), the 59 synonymous codon usage patterns of genes in the leading strand are highly similar to those in the lagging strand. The over-represented codons and the under-represented codons were identified. A comparison of the synonymous codon usage pattern of M. bovis and cattle (susceptible host) indicated the independent formation of synonymous codon usage of M. bovis. Principal component analysis revealed that (i) strand-specific mutational bias fails to affect the synonymous codon usage pattern in the leading and lagging strands, (ii) mutation pressure from nucleotide content plays a role in shaping the overall codon usage, and (iii) the major trend of synonymous codon usage has a significant correlation with the gene expression level that is estimated by the codon adaptation index. The plot of the effective number of codons against the G+C content at the third codon position also reveals that mutation pressure undoubtedly contributes to the synonymous codon usage pattern of M. bovis. Additionally, the formation of the overall codon usage is determined by certain evolutionary selections for gene function classification (30S protein, 50S protein, transposase, membrane protein, and lipoprotein) and translation elongation region of genes in M. bovis. The information could be helpful in further investigations of evolutionary mechanisms of the Mycoplasma family and heterologous expression of its functionally important proteins.     

Regularities of the nucleotide sequence at the 5'-end of the codon in Escherichia coli genes

The frequencies of occurrence of nucleotides at the 5' side of codons have been determined in highly and weakly expressed genes from E. coli. Significant constraints on the nucleotide 5' to some codons were found in highly expressed genes. Certain rules of synonymous codon usage depending on the amino acid 3' of the codon were established. E. g., codon possessing quanosine in the third position (NNG) are preferred over NNA if the next amino acid is lysine (P less than 10(-5)). On the other hand, rules of synonymous codon usage in relation to 5' flanking nucleotide were found. For example, when coding for aspartic acid, GAC codon is preferred over GAU (P less than 0.001) if uridine is 5' to codon and on the contrary GAU is favoured (P less than 0.0001) if quanosine is at the 5' side of aspartic acid codon. These rules can be used in the chemical synthesis of genes designed for expression in E. coli.     

同义密码子携带多少蛋白质二级结构信息

应用信息论方法考察了大肠杆菌人两种生物的同义密码子用语和蛋白质二级结构的关联情况。研究结果表明：大肠杆菌和人的基因组中都存在着一些同义密码子明显携带有蛋白质二级结构信息,尽管这些信息量都很小;同义密码子与蛋白质二级结构的关联是种属特异性。     

The relationship between synonymous codon usage and protein structure in Escherichia coli and Homo sapiens

The role of silent position in the codon on the protein structure is an interesting and yet unclear problem. In this paper, 563 Homo sapiens genes and 417 Escherichia coli genes coding for proteins with four different folding types have been analyzed using variance analysis, a multivariate analysis method newly used in codon usage analysis, to find the correlation between amino acid composition, synonymous codon, and protein structure in different organisms. It has been found that in E. coli, both amino acid compositions in differently folded proteins and synonymous codon usage in different gene classes coding for differently folded proteins are significantly different. It was also found that only amino acid composition is different in different protein classes in H. sapiens. There is no universal correlation between synonymous codon usage and protein structure in these two different organisms. Further analysis has shown that GC content on the second codon position can distinguish coding genes for different folded proteins in both organisms.     

Synonymous codon usage in different protein secondary structural classes of human genes: implication for increased non-randomness of GC3 rich genes towards protein stability

The relationship between the synonymous codon usage and different protein secondary structural classes were investigated using 401 Homo sapiens proteins extracted from Protein Data Bank (PDB). A simple Chi-square test was used to assess the significance of deviation of the observed and expected frequencies of 59 codons at the level of individual synonymous families in the four different protein secondary structural classes. It was observed that synonymous codon families show non-randomness in codon usage in four different secondary structural classes. However,when the genes were classified according to their GC3 levels there was an increase in non-randomness in high GC3 group of genes. The non-randomness in codon usage was further tested among the same protein secondary structures belonging to four different protein folding classes of high GC3 group of genes. The results show that in each of the protein secondary structural unit there exist some synonymous family that shows class specific codon-usage pattern. Moreover, there is an increased non-random behaviour of synonymous codons in sheet structure of all secondary structural classes in high GC3 group of genes. Biological implications of these results have been discussed.     

Natural selection versus primitive gene structure as determinant of codon usage

Different codons are not utilized equally in known gene sequences. One of the important biases of codon usage is observed in the form of an enrichment of RNY codons, especially within RNN codon families. Such biases could represent the residue of a primitive repeating-RNY gene structure, or the outcome of natural selection, or both. Analyses based on the rates of silent substitutions, the frequencies of base doublets, and synonymous codon ratios for Escherichia coli, yeast, Drosophila and Xenopus proteins have been performed. The results rule out any significant support for a primitive repeating-RNY or repeating-RRY gene structure, and establish the important role of natural selection in determining the choice of codons. With strong intervention by natural selection, the relationship between primitive gene structure and codon usage necessarily becomes minimal.     

Restructuring the translation initiation region of the human parathyroid hormone gene for improved expression in Escherichia coli

Overexpression of native human parathyroid hormone in Escherichia coli was achieved by a modification of the 5' end of the genomic gene sequence, thereby adapting this part of the translation initiation region to the bacterial host. Some simple rules abstracted from optimization studies of translation initiation of a beta-interferon gene were applied. These included (a) extending complementarity of the mRNA to the anticodon loop of tRNAfMet by use of a codon with a purine nucleotide directly following the ATG, (b) avoidance of stable secondary structure in the mRNA by use of synonymous A/U-rich codons, (c) elimination of a potential second Shine-Dalgarno sequence. The appropriate silent changes led to a 20-fold increase in parathyroid hormone production resulting in 4.3% of total soluble protein. This result proves the validity of our simple approach for optimization of foreign gene expression in E. coli.     

Ribosome-mediated translational pause and protein domain organization.

Because regions on the messenger ribonucleic acid differ in the rate at which they are translated by the ribosome and because proteins can fold cotranslationally on the ribosome, a question arises as to whether the kinetics of translation influence the folding events in the growing nascent polypeptide chain. Translationally slow regions were identified on mRNAs for a set of 37 multidomain proteins from Escherichia coli with known three-dimensional structures. The frequencies of individual codons in mRNAs of highly expressed genes from E. coli were taken as a measure of codon translation speed. Analysis of codon usage in slow regions showed a consistency with the experimentally determined translation rates of codons; abundant codons that are translated with faster speeds compared with their synonymous codons were found to be avoided; rare codons that are translated at an unexpectedly higher rate were also found to be avoided in slow regions. The statistical significance of the occurrence of such slow regions on mRNA spans corresponding to the oligopeptide domain termini and linking regions on the encoded proteins was assessed. The amino acid type and the solvent accessibility of the residues coded by such slow regions were also examined. The results indicated that protein domain boundaries that mark higher-order structural organization are largely coded by translationally slow regions on the RNA and are composed of such amino acids that are stickier to the ribosome channel through which the synthesized polypeptide chain emerges into the cytoplasm. The translationally slow nucleotide regions on mRNA possess the potential to form hairpin secondary structures and such structures could further slow the movement of ribosome. The results point to an intriguing correlation between protein synthesis machinery and in vivo protein folding. Examination of available mutagenic data indicated that the effects of some of the reported mutations were consistent with our hypothesis.