重组人血小板生成素在大肠杆菌中表达的研究

采用化学法全合成了编码人血小板生成素（ｔｈｒｏｍｂｏｐｏｉｅｔｉｎ,ＴＰＯ）成熟肽Ｎ端１５３氨基酸的基因序列,构建基于该合成基因的表达质粒,结果以谷胱甘肽转硫酶－ＴＰＯ１５３（ＧＳＴ－ＴＰＯ１５３）融合蛋白的方式获得了占全菌蛋白４０％的高效表达．进一步采用ＰＣＲ方法分别对ＴＰＯ合成基因及ＴＰＯｃＤＮＡ的翻译起始区（ＴＩＲ）序列进行定点突变,以降低这一区域的Ｇ－Ｃ含量．将突变序列分别插入到ｐＢＶ２２０表达载体中,重组质粒在转化大肠杆菌ＪＭ１０９后,均获得了表达,其中ＴＩＲ区突变后的合成基因表达产物约占全菌蛋白的１５％．为研究基因下游结构对表达的影响,在不改变氨基酸组成的基础上,构建了ＴＰＯ合成基因与ＴＰＯｃＤＮＡ的杂合序列表达质粒．研究结果表明翻译起始效率是影响ｒｈ－ＴＰＯ在大肠杆菌中表达的重要因素之一,同时基因下游序列的组成对表达水平也会产生影响．     

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.     

RNA primary sequence or secondary structure in the translational initiation region controls expression of two variant interferon-beta genes in Escherichia coli

Efficient expression in Escherichia coli (E. coli) of the human interferon-beta gene (IFN-beta) gene and of a chemically synthesized IFN-beta gene variant (506 base pairs; synIFN-beta) adapted to the E. coli codon usage, both fused to the E. coli atpE ribosome-binding site, is controlled either by primary sequence or by mRNA secondary-structure in the translational initiation region. High level expression of the natural human atpE/IFN-beta gene fusion is governed by the nucleotide composition preceding the initiator codon AUG. A single U----C exchange in the -2 or -1 position preceding the initiator codon AUG reduces the translational efficiency from 18% of total cellular protein to only 8% or 4%, respectively, while both U----C substitutions reduce IFN-beta expression below 1%. These sequence alterations interfere with efficient ribosome binding as revealed by toeprinting. They provide further evidence for the influence of the anticodon-flanking regions of tRNA(fMet) upon the initiation rate of translation. In contrast, translation of the synthetic variant atpE/synIFN-beta gene fusion is controlled by a moderately stable stem-loop structure (delta G = -4 kcal/mol; 37 degrees C) located within the coding region and overlapping the 30 S ribosomal subunit attachment site. That the stability of the hairpin interferes with the initiation of translation is inferred from site-directed mutagenesis and toeprint analyses. mRNA half-life in these variants is positively correlated with the rate of translation and involves two major endonucleolytic cleavage site 5'-upstream of the Shine-Dalgarno region.     

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.     

Inability of Prevotella bryantii to form a functional Shine-Dalgarno interaction reflects unique evolution of ribosome binding sites in Bacteroidetes

The Shine-Dalgarno (SD) sequence is a key element directing the translation to initiate at the authentic start codons and also enabling translation initiation to proceed in 5' untranslated mRNA regions (5'-UTRs) containing moderately strong secondary structures. Bioinformatic analysis of almost forty genomes from the major bacterial phylum Bacteroidetes revealed, however, a general absence of SD sequence, drop in GC content and consequently reduced tendency to form secondary structures in 5'-UTRs. The experiments using the Prevotella bryantii TC1-1 expression system were in agreement with these findings: neither addition nor omission of SD sequence in the unstructured 5'-UTR affected the level of the reporter protein, non-specific nuclease NucB. Further, NucB level in P. bryantii TC1-1, contrary to hMGFP level in Escherichia coli, was five times lower when SD sequence formed part of the secondary structure with a folding energy -5,2 kcal/mol. Also, the extended SD sequences did not affect protein levels as in E. coli. It seems therefore that a functional SD interaction does not take place during the translation initiation in P. bryanttii TC1-1 and possibly other members of phylum Bacteroidetes although the anti SD sequence is present in 16S rRNA genes of their genomes. We thus propose that in the absence of the SD sequence interaction, the selection of genuine start codons in Bacteroidetes is accomplished by binding of ribosomal protein S1 to unstructured 5'-UTR as opposed to coding region which is inaccessible due to mRNA secondary structure. Additionally, we found that sequence logos of region preceding the start codons may be used as taxonomical markers. Depending on whether complete sequence logo or only part of it, such as information content and base proportion at specific positions, is used, bacterial genera or families and in some cases even bacterial phyla can be distinguished.     

Translation of an atypical human cDNA requires fidelity of apurine-pyrimidine repeat region and recoding

Gain or loss of Migration inducting gene-7 (Mig-7) protein expression functional studies suggest it causes aggressive tumor cell invasion and tumor cell vessel-like structure formation. In addition, Mig-7 expression is apparently carcinoma and trophoblast cell-specific. Mig-7 is an example of an atypical gene that is unique in its induction, translation and apparent carcinoma-specific expression. However, studies of this predominantly integral membrane protein are hampered because of the cloning and expression techniques required for detection of Mig-7 protein. Because the encoding region possesses stop codons, repeat sequences and secondary structure, we hypothesized that genetically engineered E. coli are required to maintain the number of purine-pyrimidine repeats and reading frame when producing expression plasmids containing the Mig-7 sequence. Cloning Mig-7 sequence using E. coli genetically engineered to lack recombination and rearrangement capabilities prevented extension of the repeat region. Because of multiple stop codons in the sequence, three different constructs starting from three different reading frame ATG sites were tested for protein production in a human carcinoma cell line. Mig-7 protein of ~23 kD is produced from Mig-7 cDNA that contains multiple stop codons downstream from the ATG in a Kozak consensus sequence. In silico analyses imply that multiple Mig-7 mRNA secondary structures may cause frameshifting, read-through, and/or recoding of the multiple stop codons. Experimental results support that one or more of these translational events take place. In this report, we detail requirements for cloning and expression of this novel, atypical, human gene. These techniques can be used to express this unique protein for further studies.     

Mutations affecting pseudoknot control of the replication of B group plasmids.

The translational initiation region of the mRNA for the replication initiation protein (RepA) of pMU720 is predicted to be sequestered in an inhibitory secondary structure designated stem-loop III. Activation of repA translation requires both the disruption of stem-loop III by ribosomes involved in the translation and termination of the leader peptide RepB and the formation of a pseudoknot, a tertiary RNA structure. Disruption of stem-loop III by site-directed mutagenesis was found to be insufficient to allow high repA expression in the absence of pseudoknot formation, indicating that the pseudoknot acts as an enhancer of repA translation. Furthermore, extending the length of the leader peptide RepB and changing the distance between the pseudoknot and repA Shine-Dalgarno sequence were found to have major effects on the translation of repA.     

Nucleotide and deduced amino acid sequence of the gene for a novel protein with a possible regulatory function encoded in the β operon of Staphylococcus aureus

Abstract Although considerable homology exists between the translation products of the rplL, rpoB and rpoC genes of the β operons of the Gram-negative organism Escherichia coli and the Gram-positive Staphylococcus aureus the region between the rplL and rpoB genes is quite different in the two bacterial species. In E. coli the 324 bp has three centres of dyad symmetry in the first half of the sequence and multiple nonsense codons in all three reading frames. By contrast, the corresponding region in S. aureus consists of 1000 bp capable of forming a similar arrangement of stem-loop structures but with an open reading frame, sited 177 bp downstream of the end of rplL and 217 bp upstream of the beginning of the rpoB gene, with consensus initiation and termination signals, which if translated would generate a 22,665 Da protein with 202 amino acids. In view of the inability to find any significant homology with other proteins in the data bank and because the evidence suggests, as in E. coli , that the rplL-rpoB intergenic sequence is involved in regulation it is proposed that the expression product of orf202 may be a further element of control in the S. aureus β operon.     

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.     

The translation of recombinant proteins in E. coli can be improved by in silico generating and screening random libraries of a -70/+96 mRNA region with respect to the translation initiation codon

Recombinant protein translation in Escherichia coli may be limited by stable (i.e. low free energy) secondary structures in the mRNA translation initiation region. To circumvent this issue, we have set-up a computer tool called 'ExEnSo' (Expression Enhancer Software) that generates a random library of 8192 sequences, calculates the free energy of secondary structures of each sequence in the -70/+96 region (base 1 is the translation initiation codon), and then selects the sequence having the highest free energy. The software uses this 'optimized' sequence to create a 5' primer that can be used in PCR experiments to amplify the coding sequence of interest prior to sub-cloning into a prokaryotic expression vector. In this article, we report how ExEnSo was set-up and the results obtained with nine coding sequences with low expression levels in E. coli. The free energy of the -70/+96 region of all these coding sequences was increased compared to the non-optimized sequences. Moreover, the protein expression of eight out of nine of these coding sequences was increased in E. coli, indicating a good correlation between in silico and in vivo results. ExEnSo is available as a free online tool.     

Characterization of an associated microfibril protein through recombinant DNA techniques.

The complete primary structure of a new extracellular protein associated with elastic fiber microfibrils was determined by recombinant DNA techniques. Antiserum to insoluble bovine ocular zonule protein was used to screen a lambda gt11 cDNA expression library constructed from whole chick embryo poly(A)+ RNA. The cDNAs encoding immunoreactive fusion polypeptides were then used to rescreen the library by plaque hybridization. Nucleotide sequencing of overlapping cDNA clones revealed an open translation reading frame of 1326 bases beginning at an initiation start sequence and ending at a stop codon. The contiguous cDNA sequence contains a 3'-untranslated region of 563 bases with a possible polyadenylation site 16 bases upstream from the poly(A) tail. Primer extension of chick aortic mRNA taken together with the sequence data, reveals a 5'-untranslated region of 95 bases extending upstream from the translation start site. Northern blot analyses indicated that the isolated cDNA hybridized with a 2.1-kilobase mRNA in preparations of whole chick embryo and chick embryonic aortic, heart, and muscle RNAs. The initial translation protein encoded by the cDNA is 53,932 kDa and possesses a hydrophilic amino acid composition with glutamic acid comprising 22% of the total amino acid residues. Antiserum was elicited to a synthetic peptide sequence (14 amino acids) encoded within the deduced protein primary structure. Western blots of extracted proteins from chick embryonic aortae cultured in the presence of beta-aminopropionitrile showed that the medium and a mild salt extract contained an immunoreactive protein possessing an apparent molecular mass of 58,000 whereas harsh denaturants extracted a 32,000-kDa protein. Pulse-chase experiments using radiolabeled lysine showed that the newly synthesized 58,000-kDa protein was chased into a 32,000-kDa protein within a 2-24-h period. Immunoelectron microscopy of tissue sections from chick aortae, bovine nuchal ligament, and human ocular zonules showed that the peptide-elicited antibody localized specifically to ultrastructurally definable microfibril structures.     

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.     

Importance of the leader region of mRNA for translation initiation of ColE2 Rep protein

Translation initiation of mRNA encoding the Rep protein of the ColE2 plasmid required for initiation of plasmid DNA replication is fairly efficient in Escherichia coli cells despite the absence of a canonical Shine-Dalgarno sequence. To define sequences and structural elements responsible for translation efficiency of the Rep mRNA, a series of rep-lacZalpha translational fusions bearing various mutations in the region encoding the leader region of the Rep mRNA was generated and tested for the translation activity by measuring the beta-galactosidase activity. We showed that the region rich in A and U between the stem-loop II structure and GA cluster sequence, formation of the stem-loop II structure, but not its sequence, and the region between the GA cluster sequence and initiation codon are important along with the GA cluster sequence for efficient translation of the Rep protein. The existence of these important regions in the leader region of the Rep mRNA may explain the mechanism of inhibition of the Rep protein translation by an antisense RNA (RNAI), which is complementary to the leader region.     

Modelling of translation of human protein disulfide isomerase in Escherichia coli-A case study of gene optimisation

Recombinant human protein disulfide isomerase (PDI) was expressed in vivo in Escherichia coli using a non-optimised gene sequence and an optimised sequence with four 5' codons substituted by synonymous codons that take less time to translate. The optimisation resulted in a 2-fold increase of total PDI concentration and by successive optimisation with expression at low temperature in a 10-fold increase of the amount of soluble PDI in comparison with the original wild-type construct. The improvement can be due to a faster clearing of the ribosome binding site on the mRNA, elevating the translation initiation rate and resulting in higher ribosome loading and better ribosome protection of the PDI mRNA against endonucleolytic cleavage by RNase. This hypothesis was supported by a novel computer simulation model of E. coli translational ribosome traffic based upon the stochastic Gillespie algorithm. The study indicates the applicability of such models in optimisation of recombinant protein sequences.     

Bacterial expression of rat liver succinyl-CoA synthetase alpha-subunit. Factors that contribute to blocked translation of transcripts encoding a mitochondrial signal sequence

This study comprises a detailed evaluation of factors that are necessary to achieve high levels of expression of eukaryotic proteins in bacterial systems. We attempted to express a rat liver cDNA clone encoding the precursor to the alpha-subunit of succinyl-CoA synthetase in an Escherichia coli expression system, without success. Removal of the region encoding the mitochondrial signal peptide (115 nucleotides) allowed efficient expression of the mature protein. This nucleotide sequence was shown to block expression at the level of translation. Two regions within this fragment were able to block the expression of other genes such as E. coli lacZ. Inhibition of expression was due to the close proximity of these inhibitory sequences with the translation initiation region (TIR). Insertion of a spacer between the inhibitory sequence and the TIR relieved the block in translation. Analysis of the 115-nucleotide fragment identified sequences capable of extensive base-pairing with the Shine-Dalgarno and surrounding sequences. Such secondary structures are capable of blocking the formation of competent translation initiation complexes.