A Japanese encephalitis virus peptide present on Johnson grass mosaic virus-like particles induces virus-neutralizing antibodies and protects mice against lethal challenge

Protection against Japanese encephalitis virus (JEV) is antibody dependent, and neutralizing antibodies alone are sufficient to impart protection. Thus, we are aiming to develop a peptide-based vaccine against JEV by identifying JEV peptide sequences that could induce virus-neutralizing antibodies. Previously, we have synthesized large amounts of Johnson grass mosaic virus (JGMV) coat protein (CP) in Escherichia coli and have shown that it autoassembled to form virus-like particles (VLPs). The envelope (E) protein of JEV contains the virus-neutralization epitopes. Four peptides from different locations within JEV E protein were chosen, and these were fused to JGMV CP by recombinant DNA methods. The fusion protein autoassembled to form VLPs that could be purified by sucrose gradient centrifugation. Immunization of mice with the recombinant VLPs containing JEV peptide sequences induced anti-peptide and anti-JEV antibodies. A 27-amino-acid peptide containing amino acids 373 to 399 from JEV E protein, present on JGMV VLPs, induced virus-neutralizing antibodies. Importantly, these antibodies were obtained without the use of an adjuvant. The immunized mice showed significant protection against a lethal JEV challenge.     

人乳头瘤病毒18型L1蛋白在大肠埃希菌中的可溶性表达及病毒样颗粒的形成

目的利用大肠埃希菌系统可溶性表达人乳头瘤病毒18型(HPV18)L1蛋白,纯化和重组装获得HPV18病毒样颗粒(VLPs),为进一步研制HPV18基因工程疫苗奠定基础。方法首先按大肠埃希菌密码子偏好进行HPV18L1全基因合成,经PCR扩增出截短的HPV18L1基因,构建重组表达载体PET30a-L1,通过优化表达在大肠埃希菌BL21中可溶性表达L1蛋白,其次采用硫酸铵沉淀、离子交换层析、疏水层析后,获得高纯度的的L1蛋白,再通过解聚和重聚获得VLPs。结果全基因优化并截短的HPV18L1蛋白在大肠埃希菌系统中以可溶形式表达,纯化后的蛋白纯度达到90%以上,电镜下观察到直径为60 nm的VLPs颗粒。结论利用大肠埃希菌系统可溶性表达非融合HPV18L1蛋白,并获得均一的VLPs颗粒,为疫苗的开发奠定基础。     

Recombinant dengue virus type 2 envelope/hepatitis B surface antigen hybrid protein expressed in Pichia pastoris can function as a bivalent immunogen

A truncated version of the dengue virus type 2 envelope protein (Den2E) encoding the first 395 amino acid (aa) residues, and Den2E fused in-frame with the full-length 226-aa hepatitis B surface antigen (Den2E-HBsAg) protein were expressed in the methylotrophic yeast, Pichia pastoris. Both the recombinant proteins showed evidence of the capacity to form high molecular weight aggregates. Electron microscopic analysis of the purified proteins showed that while Den2E displayed an amorphous morphology, Den2E-HBsAg existed as well-structured virus-like particles (VLPs). Using immuno-gold electron microscopy, these VLPs were demonstrated to contain both components of the Den2E-HBsAg hybrid protein. Seroanalysis showed that the hybrid VLPs could function in vivo as bivalent immunogens, which could elicit immune responses directed against both components of the hybrid protein, as evidenced by ELISA, immunoprecipitation and immunofluorescence data.     

Complete nucleotide sequence of the Escherichia coli recB gene.

The complete nucleotide sequence of the Escherichia coli recB gene which encodes a subunit of the ATP-dependent DNase, Exonuclease V, has been determined. The proposed coding region for the RecB protein is 3543 nucleotides long and would encode a polypeptide of 1180 amino acids with a calculated molecular weight of 133,973. The start of the recB coding sequence overlaps the 3' end of the upstream ptr gene, and the recB termination codon overlaps the initiation codon of the downstream recD gene, suggesting that these genes may form an operon. No sequences which reasonably fit the consensus for an E. coli promoter could be identified upstream of the proposed recB translational start. The predicted RecB amino acid sequence contains regions of homology with ATPases, DNA binding proteins and DNA repair enzymes.     

人白细胞介素－3基因翻译起始区的改造提高其在大肠杆菌中的表达水平

为了提高人白细胞介素－3(bhIL-3)在大肠杆菌中的表达,在计算机辅助下,设计合成了PCR突变引物,用于改造起始密码AUG上下游序列,并在不改变5’端氨基酸编码的前提下,尽可能选用大肠杆菌高频使用的密码子。将经改造后的Hil-3cDNA和翻译起始区置于PL启动子之下,转入大肠杆菌Tapl06,经42℃热诱导后．获得表达产物,提高表达水平近一倍,表达量达到菌体总蛋白量的30％左右。表达产物经Western blot验证,经PVDF膜转移后进行N端顺序分析,证明前15个氨基酸正确,产物经包涵体纯化后,纯度提高至80％以上,初步复性后能明显促进Hil－3依赖细胞的生长。     

Potato Virus Y-Like Particles as a New Carrier for the Presentation of Foreign Protein Stretches

Virus-like particle (VLP) technology represents a promising approach for the creation of efficient vaccines and materials for use in nanotechnological applications. For construction of a new carrier for foreign protein sequences, the coat protein (CP) gene from potato virus Y (PVY) was cloned and expressed in Escherichia coli cells. The PVY CP self-assembles into PVY-like particles, as demonstrated by electron microscopy analysis of purified VLP preparations. The PVY CP with an N-terminal insertion of a foreign epitope (preS1) or of a whole protein (rubredoxin) retains its ability to form filamentous particles, whereas adding a foreign sequence to the C-terminus of the PVY CP generates mostly unstructured protein aggregates. This new filamentous plant virus-derived VLP carrier accommodates a foreign protein sequence that is up to 71 amino acids in length on the VLP surface and can be produced in E. coli in preparative amounts. The PVY CP VLPs are stable in physiological conditions, but they are sensitive to EDTA, high salt, and extreme pH. The presence of the preS1 epitope decreases the stability of the chimeric PVY CP particles at elevated temperatures. Mice that are immunized with chimeric PVY CP particles carrying preS1 epitopes exhibit a strong anti-preS1 immune response, even in the absence of adjuvants.     

Characterization of porcine circovirus type 2 (PCV2) capsid particle assembly and its application to virus-like particle vaccine development

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. The sole structural capsid protein of PCV2, Cap, consists of major antigenic domains, but little is known about the assembly of capsid particles. The purpose of this study is to produce a large amount of Cap protein using Escherichia coli expression system for further studying the essential sequences contributing to formation of particles. By using codon optimization of rare arginine codons near the 5'-end of the cap gene for E. coli, a full-length Cap without any fusion tag recombinant protein (Cap1-233) was expressed and proceeded to form virus-like particles (VLPs) in normal Cap appearance that resembled the authentic PCV2 capsid. The N-terminal deletion mutant (Cap51-233) deleted the nuclear localization signal (NLS) domain, while the internal deletion mutant (CapΔ51-103) deleted a likely dimerization domain that failed to form VLPs. The unique Cys108 substitution mutant (CapC/S) exhibited most irregular aggregates, and only few VLPs were formed. These results suggest that the N-terminal region within the residues 1 to 103 possessing the NLS and dimerization domains are essential for self-assembly of stable Cap VLPs, and the unique Cys108 plays an important role in the integrity of VLPs. The immunogenicity of PCV2 VLPs was further evaluated by immunization of pigs followed by challenge infection. The Cap1-233-immunized pigs demonstrated specific antibody immune responses and are prevented from PCV2 challenge, thus implying its potential use for a VLP-based PCV2 vaccine.     

Assembly of bacteriophage Qbeta virus-like particles in yeast Saccharomyces cerevisiae and Pichia pastoris

Recombinant bacteriophage Qbeta coat protein (CP), which has been proposed as a promising carrier of foreign epitopes via their incorporation either by gene engineering techniques or by chemical coupling, efficiently self-assembles into virus-like particles (VLPs) when expressed in Escherichia coli. Here, we demonstrate expression and self-assembly of Qbeta CP in yeast Saccharomyces cerevisiae and Pichia pastoris. Production reached 3-4 mg/1g of wet cells for S. cerevisiae and 4-6 mg for P. pastoris, which was about 15-20% and 20-30% of the E. coli expression level, respectively. Qbeta VLPs were easily purified by size-exclusion chromatography in both cases and contained nucleic acid, shown by native agarose gel electrophoresis. The obtained particles were highly immunogenic in mice and the resulting sera recognized both E. coli- and yeast-derived Qbeta VLPs equally well.     

Identification of cysteine-10 of protein S18 as part of the mRNA-binding site of Escherichia coli ribosomes by affinity-labeling studies with a chemically reactive A-U-G analog.

The reaction of a bromoacetamidophenyl derivative of the initiation codon A-U-G (A-U-G) with tight couples of Escherichia coli ribosomes leads to an exclusive crosslinking of label to protein S18. This crosslinking inhibits A-U-G-directed fMet-tRNAfMet binding into the puromycin-sensitive site of ribosomes and stimulates elongation-factor-dependent binding of Met-tRNAmMet. It is, therefore, concluded that protein S18 is located at or near the aminoacyl-tRNA binding site of E. coli ribosomes. Peptide as well as amino acid analysis shows that the reaction between A-U-G and ribosomes took place at cysteine-10 of protein S18. A-U-G could not be crosslinked to ribosomal proteins of the temperature-sensitive E. coli strain 258ts, where arginine-11 of protein S18 is replaced by a cysteine residue.     

同义密码子的反常蛋白质二级结构偏好性

统计分析了 119种人蛋白质和 92种大肠杆菌蛋白质的mRNA序列和蛋白质二级结构的关系 .从二肽频数出发 ,研究了同义密码子使用对蛋白质二级结构的影响 ,证明其影响在 10 %到 2 0 %的量级 .对于人和大肠杆菌 ,在 90 %置信水平上 ,4 0 0对二肽中分别有 79对和 6 0对 ,在 95 %置信水平上 ,分别有 4 5对和 36对二肽的相应密码子二联体具有不同于氨基酸的反常二级结构偏好性 ,并且这种反常不能归因于随机涨落     

The primary structure of E. coli RNA polymerase, Nucleotide sequence of the rpoC gene and amino acid sequence of the beta''-subunit.

The primary structure of the E. coli rpoC gene (5321 base pairs) coding the beta'-subunit of RNA polymerase as well as its adjacent segment have been determined. The structure analysis of the peptides obtained by cleavage of the protein with cyanogen bromide and trypsin has confirmed the amino acid sequence of the beta'-subunit deduced from the nucleotide sequence analysis. The beta'-subunit of E. coli RNA polymerase contains 1407 amino acid residues. Its translation is initiated by codon GUG and terminated by codon TAA. It has been detected that the sequence following the terminating codon is strikingly homologous to known sequences of rho-independent terminators.     

Soybean DapA mutations encoding lysine-insensitive dihydrodipicolinate synthase

In plants, the rate-limiting step in the pathway for lysine synthesis is catalyzed by the enzyme dihydrodipicolinate synthase (DS), which is encoded by the DapA gene. We previously cloned the soybean (Glycine max cv. Century) DapA gene in Escherichia coli to express functional soybean DS protein. Like the wild-type soybean DS enzyme, the DS activity encoded by the cloned gene was extremely sensitive to feedback inhibition by micromolar concentrations of lysine. Three mutants of the soybean DapA gene were constructed using PCR: one with a single amino acid substitution at codon 104, another with a single amino acid substitution at codon 112, and a mutant containing both modifications. When expressed in E. coli, the mutant DS activities were insensitive to lysine at concentrations up to 1 mM.     

TTG as the initiation codon of Salmonella slyA, a gene required for survival within macrophages.

The slyA gene, which has been implicated in the virulence of Salmonella serovar Typhimurium and its survival in macrophages, is widely distributed among different Salmonella serovars. In this study, we cloned and sequenced the translational initiation region of the slyA gene from nine different serovars and found sequence differences in the previously proposed ATG initiation codon but not in a TTG triplet, another putative initiation codon in the slyA gene. Therefore, we determined the actual translational initiation site of the slyA gene by analyzing slyA genes with defined mutation in either the ATG or TTG sequences in an in vitro translation assay and a quantitative hemolytic assay in Escherichia coli. The replacement of TTG by TTC in the slyA gene significantly reduced both the amount of protein synthesized and the hemolytic activity of a transformed strain of E. coli, while replacement of ATG by ATC had no effect in these assays. In addition, the amino acid sequence analysis of the His-tagged SlyA protein showed that it was identical with the amino acid sequence deduced from the 5' end of the slyA gene with a TTG initiation codon. Our results suggest that TTG serves as the translational initiation codon for the slyA gene of Salmonella.     

Tandem translation of Bacillus subtilis initiation factor IF2 in E. coli. Over-expression of infBB.su in E. coli and purification of alpha- and beta-forms of IF2B.su.

The protein synthesis initiation factor, IF2, in Bacillus subtilis has previously been characterized as being present in two forms, alpha and beta, of molecular mass 79 and 68 kDa, respectively, on the basis of their cross-reaction with anti-E. coli IF2 antibodies and by the DNA sequence of the gene for IF2, infBB.su. In this work we have cloned infBB.su in E. coli cells. Two proteins of molecular mass identical to the B. subtilis IF2 alpha and -beta were over-expressed and purified using a new three-step ion-exchange chromatography procedure. The N-terminal amino acid sequence of the two proteins was determined and the results confirmed that the two forms were IF2 alpha and -beta, both encoded by the infB gene. The N-terminal amino acid sequence determined for IF2 beta is Met94-Gln-Asn-Asn-Gln-Phe. The presence of methionine at position 94 shows that this form is, in fact, the result of a second translational initiation in infBB.su mRNA, since the codon at amino acid position 94 is GUG, which is the normal codon for valine, but also known to be an initiator codon. This is a new example of the unusual tandem translation in E. coli of an open mRNA reading frame.     

DNA sequence analysis of the dnaK gene of Escherichia coli B and of two dnaK genes carrying the temperature-sensitive mutations dnaK7(Ts) and dnaK756(Ts).

The DNA sequence of the dnaK gene of Escherichia coli was analyzed. The nucleotide sequence of the wild-type dnaK gene of E. coli B differed from that of E. coli K-12 in 15 bp, none of which altered the amino acid sequence. Two temperature-sensitive dnaK mutations were examined by cloning and sequence analyses. Results showed that one dnaK mutation, dnaK7(Ts), was a one-base substitution of T for C at nucleotide position 448 in the open reading frame yielding an amber nonsense codon. The other mutation, dnaK756(Ts), consisted of base substitutions (A for G) at three nucleotide positions, 95, 1364, and 1403, in the open reading frame resulting in an aspartic acid codon in place of a glycine codon.     

Influence of the second and third codon on the expression of recombinant hirudin in E. coli

The effect of all possible codons corresponding to the second and third amino acid (isoleucine and threonine) on the expression level of hirudin in E. coli has been analysed. These levels could not be correlated with changes in primary and secondary mRNA structure. A decrease in the rate of synthesis and of product accumulation follows the introduction for ile of the ATA codon which is of very low usage, and for thr of the ACC codon, which results in homology of the mRNA with the 3'-end of 16S rRNA. The results are discussed according to current concepts of protein expression in E. coli.     

Cloning of murine SeGpx cDNA and synthesis of mutated GPx proteins in Escherichia coli.

Glutathione peroxidase (GPx) of mammalian cells and Escherichia coli formate dehydrogenase both contain a selenocysteine (SeCys) in their amino acid (aa) sequence. In these two enzymes, this aa is encoded by a UGA codon, which is usually a stop codon for protein synthesis. We constructed plasmids to test the synthesis of GPx in E. coli. These constructions permitted high-level production of GPx mutants, where the SeCys codon was replaced by cysteine (UGC, UGU) or serine (UCA) codons, but synthesis of selenoprotein could not be detected: our data suggest that signals used for the recognition of the UGA codon as a SeCys codon are not conserved between E. coli and mammalian cells.     

The nature of the minimal 'selenocysteine insertion sequence' (SECIS) in Escherichia coli.

The UGA codon, usually a stop codon, can also direct the incorporation into a protein of the modified amino acid selenocysteine. This UGA decoding process requires a cis -acting mRNA element called 'selenocysteine insertion sequence' (SECIS) that can form a stem-loop structure. In Escherichia coli the SECIS of the selenoprotein formate dehydrogenase (FdhH) mRNA has been previously described to consist of at least 40 nucleotides following the UGA codon. Here we determined the nature of the minimal SECIS required for the in vivo UGA-directed selenocysteine incorporation in E.coli . Our study is based on extensive mutational analysis of the fdhF SECIS DNA located in a lac' Z fusion. We found that the whole stem-loop RNA structure of the E.coli fdhF SECIS previously described is not required for the UGA-directed selenocysteine incorporation in vivo . Rather, only its upper stem-loop structure of 17 nucleotides is necessary on the condition that it is located in a proper distance (11 nucleotides) from the UGA codon. Based on these observations, we present a new model for the minimal E.coli SECIS.