Intranasal administration of an Escherichia coli-expressed codon-optimized rotavirus VP6 protein induces protection in mice

We are developing rotavirus vaccines based on the VP6 protein of the human G1P[8] [corrected] [J. Virol. 73 (1999) 7574] CJN strain of rotavirus. One prototype candidate consisting of MBP::VP6::His6, a chimeric protein of maltose-binding protein, VP6 and hexahistidine, was expressed mainly as truncated polypeptides in Escherichia coli BL21(DE3) cells. A possible reason for this extensive truncation is the high frequencies of rare bacterial codons within the rotavirus VP6 gene. Expression of truncated recombinant VP6 was found to be reduced, and expression of complete VP6 protein was simultaneously increased, when the protein was expressed in Rosetta(DE3)pLacI E. coli cells that contain increased amounts of transfer RNAs for a selection of rare codons. The same observation was made when a synthetic codon-optimized CJN-VP6 gene was expressed in E. coli BL21 or Rosetta cells. To increase protein recovery, recombinant E. coli cells were treated with 8M urea. Denatured, full-length MBP::VP6::His6 protein was then purified and used for intranasal vaccination of BALB/c mice (2 doses administered with E. coli heat-labile toxin LT(R192G) as adjuvant). Following oral challenge with the G3P[16] [corrected] [J. Virol. 76 (2002) 560] EDIM strain of murine rotavirus, protection levels against fecal rotavirus shedding were comparable (P>0.05) between groups of mice immunized with denatured codon-optimized or native (not codon-optimized) immunogen with values ranging from 87 to 99%. These protection levels were also comparable to those found after immunization with non-denatured CJN VP6. Thus, expression of complete rotavirus VP6 protein was greatly enhanced by codon optimization, and the protection elicited was not affected by denaturation of recombinant VP6.     

Identification and quantification of N alpha-acetylated Y. pestis fusion protein F1-V expressed in Escherichia coli using LCMS E

N-terminal acetylation in E coli is a rare event catalyzed by three known N-acetyl-transferases (NATs), each having a specific ribosomal protein substrate. Multiple, gram-scale lots of recombinant F1-V, a fusion protein constructed from Y. Pestis antigens, were expressed and purified from a single stably transformed E. coli cell bank. A variant form of F1-V with mass increased by 42-43 Da was detected in all purified lots by electrospray orthogonal acceleration time-of-flight mass spectrometry (MS). Peptide mapping LCMS localized the increased mass to an N-terminal Lys-C peptide, residues 1-24, and defined it as +42.0308+/-0.0231 Da using a LockSpray exact mass feature and a leucine enkaphalin mass standard. Sequencing of the variant 1-24 peptide by LCMS and high-energy collision induced dissociation (LCMS(E)) further localized the modification to the amino terminal tri-peptide ADL and identified the modification as N(alpha)-acetylation. The average content of N(alpha)-acetylated F1-V in five lots was 24.7+/-2.6% indicating that a stable acetylation activity for F1-V was established in the E. coli expression system. Alignment of the F1-V N-terminal sequence with those of other known N(alpha)-acetylated ectopic proteins expressed in E. coli reveals a substrate motif analogous to the eukaryote NatA' acetylation pathway and distinct from endogenous E. coli NAT substrates.     

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.     

Overproduction in Escherichia coli and purification of Epstein-Barr virus EBNA-1

Epstein-Barr virus nuclear antigen 1 (EBNA-1) is a multi-functional protein of the Epstein-Barr virus (EBV). Due to its low abundance in EBV-transformed cells, overproduction in a foreign host is preferred to obtain purified EBNA-1 protein. The EBNA-1 gene possesses a large number of Escherichia coli rare codons (23%). By using E. coli BL21(DE3)Rosetta2 cells that augment the low-abundance tRNA genes, the expression level of EBNA-1 in E. coli was greatly enhanced. EBNA-1 was then purified by applying the whole cell extract soluble fraction to a Ni-NTA Superflow column and eluting with an imidazole gradient. The improved overexpression in E. coli followed by a one-step Ni-NTA purification resulted in a sufficient amount of pure EBNA-1 protein to test DNA binding activity, and prepare and test EBNA-1-specific monoclonal antibodies (mAbs).     

Production of recombinant thermostable proteins expressed in Escherichia coli: completion of protein synthesis is the bottleneck

Heterologous expression and high yield purification of proteins are frequently required for structural and functional investigations. Purification of recombinant thermostable proteins is essentially trivial since unwanted mesophilic host protein can efficiently be removed by heat denaturation. However, heterologous expression in E. coli often results in truncated protein forms. In many cases, this is a consequence of abundant codons in heterologous genes, which are decoded by rare tRNAs in E. coli-a combination that can be responsible for translational stalling and termination during protein biosynthesis. Other complications may originate from potential initiation codons and ribosomal binding sites present inside the open reading frame of the target gene or from other less well defined phenomena such as mRNA instability. Separation of full-length protein from truncated forms is a serious chromatographic problem that can be solved in the expression step. We have investigated the heterologous expression and purification of two translation initiation factors from the hyperthermophilic sulphate-reducing archaeon, Archaeoglobus fulgidus. Expression in E. coli was optimised to avoid truncated forms completely by complementation with the plasmids pSJS1244, pRIG, pCODON+ and pLysSR.A.R.E harbouring and expressing genes encoding rare tRNAs corresponding to the codons AGA, AGG, AUA, CUA, GGA, AAG and CCC. Two expression strains, C41(DE3) and C43(DE3) were found highly advantageous when combined with rare tRNA encoding plasmids as compared to BL21(DE3). We have also investigated the effects of site directed mutagenesis on rare lysine encoding AAG doublets as well as two methionine residues preceded by potential ribosomal binding sites. The expression approach presented here has enabled us to purify gram quantities of full-length protein by one step of ion-exchange chromatography and is generally applicable to many other heterologously expressed thermostable proteins.     

Expression and purification of the SsbB protein from Streptococcus pneumoniae

The Gram positive bacterium, Streptococcus pneumoniae, has two genes, designated ssbA and ssbB, which are predicted to encode single-stranded DNA binding proteins (SSB proteins). We have shown previously that the SsbA protein is similar in size and in biochemical properties to the well-characterized SSB protein from Escherichia coli. The SsbB protein, in contrast, is a smaller protein and has no counterpart in E. coli. This report describes the development of an expression system and purification procedure for the SsbB protein. The ssbB gene was amplified from genomic S. pneumoniae DNA and cloned into the E. coli expression vector, pET21a. Although, we had shown previously that the SsbA protein is strongly expressed from pET21a in the E. coli strain BL21(DE3)pLysS, no expression of the SsbB protein was detected in these cells. However, the SsbB protein was strongly expressed from pET21a in the Rosetta(DE3)pLysS strain, a derivative of BL21(DE3)pLysS which supplies the tRNAs for six codons that are used infrequently in E. coli. The differential expression of the two SSB proteins in the parent BL21(DE3)pLysS strain was apparently due to the presence of two rare codons in the ssbB gene sequence that are not present in the ssbA sequence. Using the Rosetta(DE3)pLysS/pETssbB expression system, a protocol was developed in which the SsbB protein was purified to apparent homogeneity. DNA binding assays confirmed that the purified SsbB protein had single-stranded DNA binding activity. The expression and purification procedures reported here will facilitate further investigations into the biological role of the SsbB protein.     

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.     

Mistranslational errors associated with the rare arginine codon CGG in Escherichia coli

In Escherichia coli, CGG is a rare arginine codon occurring at a frequency of 0.54% in all E. coli mRNAs or 9.8% when an arginine residue is encoded for. When present in high numbers or in clusters in highly expressed recombinant mRNA, rare codons can cause expression problems compromising product yield and translational fidelity. The coding region for an N-terminally polyhistidine tagged p27 protease domain from Herpes Simplex Virus 2 (HSV-2) contains 11 of these rare arginine codons, with 3 occurring in tandem near the C-terminus of the protein. When expressed in E. coli, the majority of the recombinant material produced had an apparent molecular mass of 31 kDa by SDS-PAGE gels or 3 kDa higher than predicted. Detailed biochemical analysis was performed on chemical and enzymatic digests of the protein and peptide fragments were characterized by Edman and MS/MS sequencing approaches. Two major species were isolated comprising +1 frameshift events at both the second and third CGG codons in the triplet cluster. Translation proceeded in the missense frame to the next termination codon. In addition, significant levels of glutamine misincorporating for arginine were discovered, suggesting second base misreading of CGG as CAG. Coexpression of the argX gene, which encodes the cognate tRNA for CGG codons, largely eliminated both the frameshift and misincorporation events, and increased expression levels of authentic product by up to 7-fold. We conclude that supplementation of the rare arginyl tRNA(CGG) levels by coexpression of the argX gene can largely alleviate the CGG codon bias present in E. coli, allowing for efficient and accurate translation of heterologous gene products.     

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.     

Myosin-like protein and actin-like protein from Escherichia coli K12 C600.

Myosin-like protein was obtained from E. coli by extraction with a sucrose solution and by precipitation with rabbit skeletal actin. The preparation of E. coli myosin-like protein looked very similar, in the sodium dodecyl sulfate-gel electrophoretic pattern, to that of rabbit skeletal myosin. The myosin-like protein was able to reversibly bind to rabbit actin. It had the activities of EDTA-, Ca-, and Mg-ATPases. The product in the EDTA-ATPase reaction catalyzed by the myosin-like protein was identified as ADP by ion exchange chromatography. The Mg-ATPase activity of E. coli myosin-like protein was activated by either rabbit actin or E. coli actin-like protein though the activation was much stronger by the latter. However, the myosin-like protein did not exhibit superprecipitation either with rabbit actin or with E. coli actin-like protein. Actin-like protein was also obtained from E. coli by essentially the same procedures as those described for preparation of rabbit skeletal actin. E. coli actin-like protein was capable of activating Mg-ATPase of rabbit myosin, and also of superprecipitation with rabbit myosin. Extraction from both the whole cells and the membrane fraction of E. coli strongly suggested that the myosin-like protein and the actin-like protein are both localized in the membrane fraction rather than in the cytoplasmic fraction.     

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.     

密码子优化的肺炎支原体P1蛋白在大肠杆菌中的克隆表达

目的:获得密码子优化的肺炎支原体P1黏附蛋白优势表位抗原基因,并在大肠杆菌中表达,为临床诊断试剂和疫苗研制打下基础。方法:采用生物信息学分析肺炎支原体P1蛋白的抗原表位,筛选特异性P1蛋白优势表位区;采用大肠杆菌优势密码子,设计上述P1蛋白优势表位基因序列;采用退火PCR技术合成上述基因,并利用载体pGEX-4T-2实现P1优势表位抗原在大肠杆菌中的表达;采用ELISA法对纯化的P1抗原活性进行测定。结果:肺炎支原体P1蛋白特异性抗原表位主要位于1154~1521 aa,获得的P1优化密码子基因平行突变37个稀有密码子和2个终止密码子;在大肠杆菌中表达的GST-P1融合蛋白的相对分子质量为65.9×103,纯化后重组抗原能与肺炎支原体感染者血清发生特异性的免疫反应。结论:采用密码子优化基因合成技术实现了肺炎支原体P1优势表位抗原在大肠杆菌中的高效表达,为肺炎支原体感染的诊断试剂研究提供了重要参考。     

Synonymous codon substitutions affect ribosome traffic and protein folding during in vitro translation

To investigate the possible influence of the local rates of translation on protein folding, 16 consecutive rare (in Escherichia coli) codons in the chloramphenicol acetyltransferase (CAT) gene have been replaced by frequent ones. Site-directed silent mutagenesis reduced the pauses in translation of CAT in E. coli S30 extract cell-free system and led to the acceleration of the overall rate of CAT protein synthesis. At the same time, the silently mutated protein (with unaltered protein sequence) synthesized in the E. coli S30 extract system was shown to possess 20% lower specific activity. The data suggest that kinetics of protein translation can affect the in vivo protein-folding pathway, leading to increased levels of protein misfolding.     

Cloning and characterization of the esp region from a dog attaching and effacing Escherichia coli strain 4221 and detection of EspB protein-binding to HEp-2 cells

The espA, espB and espD genes from enteropathogenic Escherichia coli were previously shown to be essential for triggering the signal transduction in infected host cells. We have cloned and determined the nucleotide sequences of the espA, espB and espD homologues from an E. coli strain (4221) isolated from a dog which manifested the attaching and effacing lesions in the small intestine. This strain is designated as a dog enteropathogenic E. coli. When comparing predicted amino acid sequences to those of the corresponding proteins from enteropathogenic E. coli O127, enterohemorrhagic E. coli serotype O26, enterohemorrhagic E. coli O157 and rabbit enteropathogenic E. coli, the EspADEPEC protein showed the same level of similarity (75% identity) with EspA of enteropathogenic E. coli O127 and rabbit enteropathogenic E. coli. The EspBDEPEC protein showed the highest similarity with the EspB of enteropathogenic E. coli O127 (99% identity). The EspDDEPEC protein showed 88% identity with the EspDEPEC. We constructed and purified a maltose-binding fusion protein containing the product of the entire espBDEPEC gene of the dog enteropathogenic E. coli strain 4221. Purified maltose-binding protein-EspBDEPEC fusion protein was shown to bind efficiently to HEp-2 cells in a localized fashion as shown by immunofluorescence microscopy. In addition, when the dog enteropathogenic E. coli strain 4221 was grown in tissue culture medium (DMEM) supplemented with serum, a secreted 36-kDa protein was identified by immunoblot analysis using a polyclonal antiserum against the maltose-binding protein-EspBDEPEC fusion protein.     

人B淋巴细胞刺激因子C端肽的免疫增强作用

用套式PCR从人胎脑cDNA文库中克隆了B淋巴细胞刺激因子C端肽 (C terminalpeptideofBlymphocytestimulator,C BLyS)的cDNA。在大肠杆菌BL2 1CodonPlus (DE3)RIL中以包含体形式表达了C BLyS。对包含体的复性条件进行了摸索 ,建立了C BLyS的透析复性与纯化方法。经复性和纯化的C BLyS可结合其受体B细胞成熟抗原 (Bcellmaturationantigen ,BCMA) 人IgG1Fc融合蛋白 ,刺激体外培养的小鼠脾脏细胞增殖 ,并且可明显增强小鼠对溶菌酶的免疫应答水平。     

pAM401-based shuttle vectors that enable overexpression of promoterless genes and one-step purification of tag fusion proteins directly from Enterococcus faecalis

Two novel Enterococcus faecalis-Escherichia coli shuttle vectors that utilize the promoter and ribosome binding site of bacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli and E. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless beta-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. beta-Galactosidase was expressed in E. coli and E. faecalis at levels of 10(3) and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.     

Preparation of recombinant six-histidine-tagged human LECT2, a chemotactic protein to neutrophils, in Escherichia coli

LECT2 is a chemotactic protein to neutrophils. A recombinant six-histidine-tagged human LECT2, (His)₆-LECT2, was expressed in E. coli using a pET21a(+) vector. The (His)₆-LECT2 was purified from the soluble fraction in E. coli as a single band in sodium dodesyl sulfate/polyacrylamide gel electrophoresis using three steps of column chromatography with Ni²⁺-charged nitrilo-triacetic acid (Ni-NTA) agarose, DEAE-Sepharose, and CM-Sepharose. The purified (His)₆-LECT2 was yielded with 96 μg from the soluble fraction of 1,500 ml culture of E. coli. The circular dichroism spectrum of (His)₆-LECT2 showed the folded structure, which is rich in β-sheet structure and rare in α-helix. This revised version was published online in June 2006 with corrections to the Cover Date.     

Analysis of an mRNA exhibiting anomalous translational specificity.

Gene 6 mRNA of Bacillus subtilis phage phi 29 is inefficiently translated under standard in vitro conditions by Escherichia coli, while it is efficiently translated by the in vitro system derived from B. subtilis. This is a rare example of the inability of E. coli to translate mRNA translated by B. subtilis. The ionic condition in the translation systems was the key component in the differential recognition of the gene 6 message by E. coli and B. subtilis ribosomes. Its translation by E. coli ribosomes was preferentially inhibited by moderate levels of KCl, while its translation by B. subtilis ribosomes was unaffected by these concentrations of salt. This preferential inhibition with E. coli ribosomes was observed in vitro as well as in vivo. While not influencing the general phenomenon of preferential inhibition, anion-specific effects were observed in overall protein synthesis. Glutamate and acetate promoted efficient synthesis over a broad range of concentrations, whereas chloride was inhibitory at all concentrations tested.     

High-level expression of soluble viral structural protein in Escherichia coli

Pharmaceutically relevant virus-like particles (VLPs) can potentially be manufactured cheaply and efficiently through in vitro assembly of viral structural protein in cell-free reactors, but a bottleneck for this processing route is the currently low-level expression of soluble viral protein in efficient cell factories such as Escherichia coli (E. coli). Here, we report expression levels of up to 180 mg L(-1) that are achievable from low-cell-density E. coli cultures using a simple and low cost strategy. We investigated effects of host strain, plasmid, inducer concentration, pre-induction temperature and cell density at induction with design of experiment (DOE). The statistical approach successfully identified significant effects and their interactions, and provided insights into the role of codon-usage effects in expression of viral structural protein. In particular, our results support the notion that full codon optimization may be unnecessary to improve expression of viral genes rich in E. coli rare codons; using a strategically modified host cell could provide a simpler and cheaper alternative.