Codon preference optimization increases heterologous PEDF expression

Pigment epithelium-derived factor (PEDF) is widely known for its neurotrophic and antiangiogenic functions. Efficacy studies of PEDF in animal models are limited because of poor heterologous protein yields. Here, we redesigned the human PEDF gene to preferentially match codon frequencies of E coli without altering the amino acid sequence. Following de novo synthesis, codon optimized PEDF (coPEDF) and the wtPEDF genes were cloned into pET32a containing a 5' thioredoxin sequence (Trx) and the recombinant Trx-coPEDF or Trx-wtPEDF fusion constructs expressed in native and two tRNA augmented E coli hosts - BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP, carrying extra copies of tRNAarg,ile,leu and tRNAarg,pro genes, respectively. Trx-PEDF fusion proteins were isolated using Ni-NTA metal affinity chromatography and PEDF purified after cleavage with factor Xα. Protein purity and identity were confirmed by western blot, MALDI-TOF, and UV/CD spectral analyses. Expression of the synthetic gene was ～3.4 fold greater (212.7 mg/g; 62.1 mg/g wet cells) and purified yields ～4 fold greater (41.1 mg/g; 11.3 mg/g wet cell) than wtPEDF in the native host. A small increase in expression of both genes was observed in hosts supplemented with rare tRNA genes compared to the native host but expression of coPEDF was ～3 fold greater than wtPEDF in both native and codon-bias-adjusted E coli strains. ΔGs at -3 to +50 of the Trx site of both fusion genes were -3.9 kcal/mol. Functionally, coPEDF was equally as effective as wtPEDF in reducing oxidative stress, promoting neurite outgrowth, and blocking endothelial tube formation. These findings suggest that while rare tRNA augmentation and mRNA folding energies can significantly contribute to increased protein expression, preferred codon usage, in this case, is advantageous to translational efficiency of biologically active PEDF in E coli. This strategy will undoubtedly fast forward studies to validate therapeutic utility of PEDF in vivo.     

Genes for tRNA(Arg) located in the upstream region of the Shiga toxin II operon in enterohemorrhagic Escherichia coli O157:H7.

We found two genes for tRNA(Arg) in the region upstream of genes for Shiga-like toxin type II (SLT-II) in Escherichia coli O157:H7. The two encoded forms of tRNA(Arg) recognize rare codons in E. coli K12 but these rare codons occur in the toxin genes at high frequency.     

High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product

We have observed that proteins, such as human tissue-type plasminogen activator, pro-urokinase or gp41 of human immunodeficiency virus, which have a high content of rare codons in their respective genes, are not readily expressed in Escherichia coli. Furthermore induction of these heterologous genes leads to growth inhibition and plasmid instability. Supplementation with tRNA(AGA/AGG(Arg)) by cotransfection with the dnaY gene, which supplies this minor tRNA, resulted in high-level production with greatly improved cell viability and plasmid stability.     

Rare codon clusters at 5'-end influence heterologous expression of archaeal gene in Escherichia coli

Proteins from hyperthermophilic microorganisms are attractive candidates for novel biocatalysts because of their high resistance to temperature extremes. However, archaeal genes are usually poorly expressed in Escherichia coli because of differences in codon usage. Genes from the thermoacidophilic archaea Sulfolobus solfataricus and Thermoplasma acidophilum contain high proportions of rare codons for arginine, isoleucine, and leucine, which are recognized by the tRNAs encoded by the argU, ileY, and leuW genes, respectively, and which are rarely used in E. coli. To examine the effects of these rare codons on heterologous expression, we expressed the Sso_gnaD and Tac_gnaD genes from S. solfataricus and T. acidophilum, respectively, in E. coli. The Sso_gnaD product was expressed at very low levels when the open reading frame (ORF) was cloned in pRSET and expressed in E. coli BL21(DE3), and was expressed at much higher levels in the E. coli BL21(DE3)-CodonPlus RIL strain, which contains extra copies of the argU, ileY, and leuW tRNA genes. In contrast, Tac_gnaD was expressed at similar levels in both E. coli strains. Comparison of the Sso_gnaD and Tac_gnaD gene sequences revealed that the 5'-end of the Sso_gnaD sequence was rich in AGA(arg) and ATA(Ile) codons. These codons were replaced with the codons commonly used in E. coli by polymerase chain reaction-mediated site-directed mutagenesis. The results of expression studies showed that a non-tandem repeat of rare codons is critical in the observed interference in heterologous expression of this gene. We concluded that the level of heterologous expression of Sso_gnaD in E. coli was limited by the clustering of the rare codons in the ORF, rather than on the rare codon frequency.     

Simple and efficient method for heterologous expression of clostridial proteins

Many clostridial proteins are poorly produced in Escherichia coli. It has been suggested that this phenomena is due to the fact that several types of codons common in clostridial coding sequences are rarely used in E. coli and the quantities of the corresponding tRNAs in E. coli are not sufficient to ensure efficient translation of the corresponding clostridial sequences. To address this issue, we amplified three E. coli genes, ileX, argU, and leuW, in E. coli; these genes encode tRNAs that are rarely used in E. coli (the tRNAs for the ATA, AGA, and CTA codons, respectively). Our data demonstrate that amplification of ileX dramatically increased the level of production of most of the clostridial proteins tested, while amplification of argU had a moderate effect and amplification of leuW had no effect. Thus, amplification of certain tRNA genes for rare codons in E. coli improves the expression of clostridial genes in E. coli, while amplification of other tRNAs for rare codons might not be needed for improved expression. We also show that amplification of a particular tRNA gene might have different effects on the level of protein production depending on the prevalence and relative positions of the corresponding codons in the coding sequence. Finally, we describe a novel approach for improving expression of recombinant clostridial proteins that are usually expressed at a very low level in E. coli.     

Enhanced heterologous expression of two Streptomyces griseolus cytochrome P450s and Streptomyces coelicolor ferredoxin reductase as potentially efficient hydroxylation catalysts

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.     

Codon usage in regulatory genes in Escherichia coli does not reflect selection for ''rare'' codons.

It has often been suggested that differential usage of codons recognized by rare tRNA species, i.e. "rare codons", represents an evolutionary strategy to modulate gene expression. In particular, regulatory genes are reported to have an extraordinarily high frequency of rare codons. From E. coli we have compiled codon usage data for highly expressed genes, moderately/lowly expressed genes, and regulatory genes. We have identified a clear and general trend in codon usage bias, from the very high bias seen in very highly expressed genes and attributed to selection, to a rather low bias in other genes which seems to be more influenced by mutation than by selection. There is no clear tendency for an increased frequency of rare codons in the regulatory genes, compared to a large group of other moderately/lowly expressed genes with low codon bias. From this, as well as a consideration of evolutionary rates of regulatory genes, and of experimental data on translation rates, we conclude that the pattern of synonymous codon usage in regulatory genes reflects primarily the relaxation of natural selection.     

Significance of Codon Usage and Irregularities of Rare Codon Distribution in Genes for Expression of BspLU11III Methyltransferases

Genes of adenine-specific DNA-methyltransferase M.BspLU11IIIa and cytosine-specific DNA-methyltransferase M.BspLU11IIIb of the type IIG BspLU11III restriction-modification system from the thermophilic strain Bacillus sp. LU11 were expressed in E. coli. They contain a large number of codons that are rare in E. coli and are characterized by equal values of codon adaptation index (CAI) and expression level measure (E(g)). Rare codons are either diffused (M.BspLU11IIIa) or located in clusters (M.BspLU11IIIb). The expression level of the cytosine-specific DNA-methyltransferase was increased by a factor of 7.3 and that of adenine-specific DNA only by a factor of 1.25 after introduction of the plasmid pRARE supplying tRNA genes for six rare codons in E. coli. It can be assumed that the plasmid supplying minor tRNAs can strongly increase the expression level of only genes with cluster distribution of rare codons. Using heparin-Sepharose and phosphocellulose chromatography and gel filtration on Sephadex G-75 both DNA-methyltransferases were isolated as electrophoretically homogeneous proteins (according to the results of SDS-PAGE).     

High level, context dependent misincorporation of lysine for arginine in Saccharomyces cerevisiae a1 homeodomain expressed in Escherichia coli.

The Saccharomyces cerevisiae a1 homeodomain is expressed as a soluble protein in Escherichia coli when cultured in minimal medium. Nuclear magnetic resonance (NMR) spectra of previously prepared a1 homeodomain samples contained a subset of doubled and broadened resonances. Mass spectroscopic and NMR analysis demonstrates that the heterogeneity is largely due to a lysine misincorporation at the arginine (Arg) 115 site. Arg 115 is coded by the 5'-AGA-3' sequence, which is quite rare in E. coli genes. Lower level mistranslation at three other rare arginine codons also occurs. The percentage of lysine for arginine misincorporation in a1 homeodomain production is dependent on media composition. The dnaY gene, which encodes the rare 5'-AGA-3' tRNA(ARG), was co-expressed in E. coli with the a1-encoding plasmid to produce a homogeneous recombinant a1 homeodomain. Co-expression of the dnaY gene completely blocks mistranslation of arginine to lysine during a1 overexpression in minimal media, and homogeneous protein is produced.     

High level expression of Thermococcus litoralis 4-alpha-glucanotransferase in a soluble form in Escherichia coli with a novel expression system involving minor arginine tRNAs and GroELS.

The Thermococcus litoralis 4-alpha-glucanotransferase (GTase) gene has a high content of AGA and AGG codons for arginine, which are extremely rare in Escherichia coli. Expression of the GTase gene in E. coli resulted in low protein production and the accumulation of inclusion bodies. However, simultaneous expression of GTase with tRNA(AGA), tRNA(AGG) and GroELS affected both the production and solubility of GTase, and production of soluble GTase increasing about 5-fold. This new E. coli expression system should be applicable to the expression of not only archaeal but also eukaryotic genes, which usually contain a large number of AGA and AGG codons.     

Close linkage of the genes serC (for phosphohydroxy pyruvate transaminase) and serS (for seryl-transfer ribonucleic acid synthetase) in Escherichia coli K-12

Escherichia coli strain K28, isolated after nitrosoguanidine mutagenesis, was found to be auxotrophic for serine. It was also temperature sensitive for growth as a result of producing an altered seryl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.11, l-serine: tRNA ligase [AMP]). The auxotrophy was caused by a mutation in the structural gene for phosphohydroxy-pyruvate transaminase (serC), which was distinct from, but closely linked to, the structural gene for seryl-tRNA synthetase (serS). We conclude that the relevant genes are in the order gal-serS-serC-aroA.     

Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli.

In Escherichia coli, the isoleucine codon AUA occurs at a frequency of about 0.4% and is the fifth rarest codon in E. coli mRNA. Since there is a correlation between the frequency of codon usage and the level of its cognate tRNA, translational problems might be expected when the mRNA contains high levels of AUA codons. When a hemagglutinin from the influenza virus, a 304-amino-acid protein with 12 (3.9%) AUA codons and 1 tandem codon, and a mupirocin-resistant isoleucyl tRNA synthetase, a 1,024-amino-acid protein, with 33 (3.2%) AUA codons and 2 tandem codons, were expressed in E. coli, product accumulation was highly variable and dependent to some degree on the growth medium. In rich medium, the flu antigen represented about 16% of total cell protein, whereas in minimal medium, it was only 2 to 3% of total cell protein. In the presence of the cloned ileX, which encodes the cognate tRNA for AUA, however, the antigen was 25 to 30% of total cell protein in cells grown in minimal medium. Alternatively, the isoleucyl tRNA synthetase did not accumulate to detectable levels in cells grown in Luria broth unless the ileX tRNA was coexpressed when it accounted for 7 to 9% of total cell protein. These results indicate that the rare isoleucine AUA codon, like the rare arginine codons AGG and AGA, can interfere with the efficient expression of cloned proteins.     

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).     

真核低分子量尿激酶原突变体基因在大肠杆菌中的高效表达

 通过一种融合抗栓肽的低分子量尿激酶原的突变体 (DscuPA 32K)在大肠杆菌中表达的研究 ,进行了一系列不同条件下的实验 .DscuPA 32K在菌株BL2 1中的表达很低 ,表达量仅为 3% ;为提高其表达量 ,引进一种整合稀有tRNA基因的菌株BL2 1 CodonPlusTM RIL ,增加大肠杆菌中识别稀有密码子的tRNA的数量 ,DscuPA 32K的表达水平确实有了很大提高 ,最大表达量约占 2 0 % .结果表明 ,富含稀有密码子的DscuPA 32K在大肠杆菌中表达受限制的因素 ,完全可以由增加稀有tRNA的数量来克服 .免疫印迹分析DscuPA 32K具有良好的抗原性 .此表达菌株可能有利于含大肠杆菌稀有密码子的真核基因在大肠杆菌中的表达 .