Expression of tetanus toxin fragment C in E. coli: high level expression by removing rare codons.

Tetanus toxin fragment C had been previously expressed in Escherichia coli at 3-4% cell protein. The codon bias for tetanus toxin in Clostridium tetani is very different from that of highly expressed homologous genes in E. coli, resulting in the presence of many rare E. coli codons in the sequence encoding fragment C. We have replaced the coding sequence by sequence optimized for codon usage in E. coli, and show that the expression of fragment C is increased. Although the level of mRNA also increased this appeared to be a secondary consequence of more efficient translation. Complete sequence replacement increased expression to approximately 11-14% cell protein but only after the promoter strength had been improved.     

Translational features of human alpha 2b interferon production in Escherichia coli

The yield of human alpha 2b interferon in Escherichia coli was optimized by replacement of low-usage arginine codons located in the mRNA 5' end. The differences observed among the various gene variants suggest that codon usage, Shine-Dalgarno-like sequences, and mRNA secondary structure contribute to the performance of E. coli translation machinery.     

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.     

Comparative nucleotide sequence analysis of growth-rate-regulated gnd alleles from natural isolates of Escherichia coli and from Salmonella typhimurium LT-2.

A comparative study of gnd genes from Escherichia coli strains isolated from natural populations and laboratory strains and from Salmonella typhimurium was undertaken. In the accompanying paper (G. J. Barcak and R. E. Wolf, Jr., J. Bacteriol. 170:365-371, 1988), we showed that the growth-rate-dependent regulation of gnd expression was conserved among four natural E. coli isolates and E. coli B/r in a manner qualitatively similar to that of the gene from E. coli K-12. Here, we report the DNA sequence of the 5' regulatory region and the first 125 codons of the structural gene for the five E. coli gnd genes and the gnd gene from S. typhimurium LT-2. The sequences differed from one another by 5% on the average. All sequences defined putative secondary structures of the mRNA leader, which were previously proposed to be important in the regulation of the K-12 gene. In addition, a sequence between codons 69 and 74, which is highly complementary to the ribosome-binding site of the mRNA, was conserved in all the genes. The sequence data are discussed with respect to potential regulatory consequences.     

Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host

Synonymous codon replacement can change protein structure and function, indicating that protein structure depends on DNA sequence. During heterologous protein expression, low expression or formation of insoluble aggregates may be attributable to differences in synonymous codon usage between expression and natural hosts. This discordance may be particularly important during translation of the domain boundaries (link/end segments) that separate elements of higher ordered structure. Within such regions, ribosomal progression slows as the ribosome encounters clusters of infrequently used codons that preferentially encode a subset of amino acids. To replicate the modulation of such localized translation rates during heterologous expression, we used known relationships between codon usage frequencies and secondary protein structure to develop an algorithm ("codon harmonization") for identifying regions of slowly translated mRNA that are putatively associated with link/end segments. It then recommends synonymous replacement codons having usage frequencies in the heterologous expression host that are less than or equal to the usage frequencies of native codons in the native expression host. For protein regions other than these putative link/end segments, it recommends synonymous substitutions with codons having usage frequencies matched as nearly as possible to the native expression system. Previous application of this algorithm facilitated E. coli expression, manufacture and testing of two Plasmodium falciparum vaccine candidates. Here we describe the algorithm in detail and apply it to E. coli expression of three additional P. falciparum proteins. Expression of the "recoded" genes exceeded that of the native genes by 4- to 1,000-fold, representing levels suitable for vaccine manufacture. The proteins were soluble and reacted with a variety of functional conformation-specific mAbs suggesting that they were folded properly and had assumed native conformation. Codon harmonization may further provide a general strategy for improving the expression of soluble functional proteins during heterologous expression in hosts other than E. coli.     

同义密码子用语的位置依赖

研究了在大肠杆菌编码区不同位置上的同底密码子用语,发现许多氨基酸的密码子用语在转译起始区有显著的变化,仅有少数氨基酸在转译区有较弱的变化,由于密码子用语与基因表达关系密切。这些结果与实验发现的编码区5‘端密码子用对表达的重要性是一致的。更进一步的结果还暗示了哪些密码子在特定位置的使用可能会影响基因表达。     

Reduced synonymous substitution rate at the start of enterobacterial genes.

Synonymous codon usage is less biased at the start of Escherichia coli genes than elsewhere. The rate of synonymous substitution between E.coli and Salmonella typhimurium is substantially reduced near the start of the gene, which suggests the presence of an additional selection pressure which competes with the selection for codons which are most rapidly translated. Possible competing sources of selection are the presence of secondary ribosome binding sites downstream from the start codon, the avoidance of mRNA secondary structure near the start of the gene and the use of sub-optimal codons to regulate gene expression. We provide evidence against the last of these possibilities. We also show that there is a decrease in the frequency of A, and an increase in the frequency of G along the E.coli genes at all three codon positions. We argue that these results are most consistent with selection to avoid mRNA secondary structure.     

Codon bias at the 3'-side of the initiation codon is correlated with translation initiation efficiency in Escherichia coli

The codon that follows the AUG initiation triplet (+2 codon) affects gene expression in Escherichia coli. We have extended this analysis using two model genes lacking any apparent Shine-Dalgarno sequence. Depending on the identity of the +2 codon a difference in gene expression up to 20-fold could be obtained. The effects did not correlate with the levels of intracellular pools of cognate tRNA for the +2 codon, with putative secondary mRNA structures, or with mRNA stability. However, most +2 iso-codons that were decoded by the same species of tRNA gave pairwise similar effects, suggesting that the effect on gene expression was associated with the decoding tRNA. High adenine content of the +2 codon was associated with high gene expression. Of the fourteen +2 codons that mediated the highest efficiency, all except two had an adenine as the first base of the codon. Analysis of the 3540 E. coli genes from the TransTerm database revealed that codons associated with high gene expression in the two expression systems are over-represented at the +2 position in natural genes. Codons that are associated with low gene expression are under-represented. The data suggest that evolution has favored codons at the +2 position that give high translation initiation.     

Analysis of the primary structure of mRNA from Escherichia coli: occurrence of nucleotides on the 3'-side of the codon

The occurrence of nucleotides of the 3' side of codons has been determined in highly and weakly expressed genes from Escherichia coli. It was found that the usage of some amino acid codons in highly expressed genes was site specific, depending on the base 3' to the codon. The role of the 3' nucleotide as a modulator of codon translation effectiveness is discussed. The rules of synonymous codon usage in relation to the 3' flanking nucleotide have been established for highly expressed genes. For example, if a triplet next to the lysine codon starts with guanosine, lysine is preferably encoded by AAA and not by AAG (P less than 10(-8), while of cytidine is 3' to the lysine codon, AAG is preferred over AAA (P less than 0.001). These rules are observed in highly and absent in weakly expressed mRNAs and can be used in the chemical synthesis of genes designed for expression in E. coli.     

Translation of chloroplast-encoded mRNA: potential initiation and termination signals.

A survey of 196 protein-coding chloroplast DNA sequences demonstrated the preference for AUG and UAA codons for initiation and termination of translation, respectively. As in prokaryotes at every nucleotide position from -25 to +25 (AUG is +1 to +3) and for 25 nucleotides 5' and 3' to the termination codon an A or U is predominant, except for C at +5 and G at +22. A Shine-Dalgarno (SD) sequence (GGAGG or tri- or tetranucleotide variant) was found within 100 bp 5' to the AUG codon in 92% of the genes. In 40% of these cases, the location of the SD sequence was similar to that of the consensus for prokaryotes (-12 to -7 5' to AUG), presumed to be optimal for translation initiation. A SD sequence could not be located in 6% of the chloroplast sequences. We propose that mRNA secondary structures may be required for the relocation of a distal SD sequences to within the optimal region (-12 to -7) for initiation of translation. We further suggest that termination at UGA codons in chloroplast genes may occur by a mechanism, involving 16S rRNA secondary structure, which has been proposed for UGA termination in E. coli.     

Experimental confirmation of a key role for non-optimal codons in protein export

Non-optimal codons are defined by low usage and low abundance of corresponding tRNA, and have an established role in translational pausing to allow the correct folding of proteins. Our previous work reported a striking abundance of non-optimal codons in the signal sequences of secretory proteins exported via the sec-dependent pathway in Escherichia coli. In the current study the signal sequence of maltose-binding protein (MBP) was altered so that non-optimal codons were substituted with the most optimal codon from their synonymous codon family. The expression of MBP from the optimized allele (malE-opt) was significantly less than wild-type malE. Expression of MBP from malE-opt was partially restored in a range of cytoplasmic and periplasmic protease deficient strains, confirming that reduced expression of MBP in malE-opt was due to its preferential degradation by cytoplasmic and periplasmic proteases. These data confirm a novel role for non-optimal codon usage in secretion by slowing the rate of translation across the N-terminal signal sequence to facilitate proper folding of the secreted protein.     

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.     

High-temperature cultivation and 5' mRNA optimization are key factors for the efficient overexpression of thermostable Deinococcus geothermalis purine nucleoside phosphorylase in Escherichia coli

Overexpression of genes from thermophiles in Escherichia coli is an attractive approach towards the large-scale production of thermostable biocatalysts. However, various factors can challenge efficient heterologous protein expression--one example is the formation of stable 5' mRNA secondary structures that can impede an efficient translation initiation. In this work, we describe the expression optimization of purine nucleoside phosphorylase from the thermophilic microbe Deinococcus geothermalis in E. coli. Poor expression levels caused by stable secondary 5' mRNA structure formation were addressed by two different approaches: (i) increasing the cultivation temperature above the range used typically for recombinant protein expression and (ii) optimizing the 5' mRNA sequence for reduced secondary structures in the translation initiation region. The increase of the cultivation temperature from 30°C to 42°C allowed a more than 10-fold increase of activity per cell and optimizing the 5' mRNA gene sequence further increased the activity per cell 1.7-fold at 42°C. Thus, the combination of high-temperature cultivation and 5' sequence optimization is described as an effective approach to overcome poor expression levels resulting from stable secondary 5' mRNA structure formation. We suggest that this method is especially suitable for improving the expression of proteins derived from thermophiles in E. coli.     

HIV-1 gag expression is quantitatively dependent on the ratio of native and optimized codons

There is a significant variation of codon usage bias among different species and even among genes within the same organisms. Codon optimization, this is, gene redesigning with the use of codons preferred for the specific expression system, results in improved expression of heterologous genes in bacteria, plants, yeast, mammalian cells, and transgenic animals. The mechanisms preventing expression of genes with rare or low-usage codons at adequate levels are not completely elucidated. Human immunodeficiency virus (HIV) represents an interesting model for studying how differences in codon usage affect gene expression in heterologous systems. Construction of synthetic genes with optimized codons demonstrated that the codon-usage effects might be a major impediment to the efficient expression of HIV gag/pol and env gene products in mammalian cells. According to another hypothesis, the poor expression of HIV structural proteins even without HIV context is attributed to the so-called cis-acting inhibitory elements (INS), which are located within the protein-coding region. They consist of AU-rich sequences and may be inactivated through the introduction of multiple mutations over the large regions of gag gene. In our work, we evaluated expression of hybrid HIV-1 gag mRNAs where wild-type (A-rich) gag sequences were combined with artificial sequences. In such "humanized" gag fragments with adapted codon usage, AT-content was significantly reduced in favor of G and C nucleotides without any changes in protein sequence. We show that wild-type gag sequences negatively influence expression of gag-reporter, and the addition of fragments with optimized codons to gag mRNA partially rescues its expression. The results demonstrate that the expression of HIV-1 gag is determined by the ratio of optimized and rare codons within mRNA. Our data also indicates that some wtgag fragments counteract the influence of the other wtgag sequences, which cause the inhibition of gag expression. The presented data do not contradict the concept of INS; yet, it makes the definition of INS more complex. This supports the idea of a broader role of the selected codon usage in influencing the expression of HIV proteins in mammalian cells.     

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

We determined the in vivo translational efficiency of 'unleadered' lacZ compared with a conventionally leadered lacZ with and without a Shine–Dalgarno (SD) sequence in Escherichia coli and found that changing the SD sequence of leadered lacZ from the consensus 5'-AGGA-3' to 5'-UUUU-3' results in a 15-fold reduction in translational efficiency; however, removing the leader altogether results in only a twofold reduction. An increase in translation coincident with the removal of the leader lacking a SD sequence suggests the existence of stronger or novel translational signals within the coding sequence in the absence of the leader. We examined, therefore, a change in the translational signals provided by altering the AUG initiation codon to other naturally occurring initiation codons (GUG, UUG, CUG) in the presence and absence of a leader and find that mRNAs lacking leader sequences are dependent upon an AUG initiation codon, whereas leadered mRNAs are not. This suggests that mRNAs lacking leader sequences are either more dependent on perfect codon–anticodon complementarity or require an AUG initiation codon in a sequence-specific manner to form productive initiation complexes. A mutant initiator tRNA with compensating anticodon mutations restored expression of leadered, but not unleadered, mRNAs with UAG start codons, indicating that codon–anticodon complementarity was insufficient for the translation of mRNA lacking leader sequences. These data suggest that a cognate AUG initiation codon specifically serves as a stronger and different translational signal in the absence of an untranslated leader.