Rubella E1 Research Articles

Designing next generation recombinant protein expression platforms by modulating the cellular stress response in Escherichia coli

BackgroundA cellular stress response (CSR) is triggered upon recombinant protein synthesis which acts as a global feedback regulator of protein expression. To remove this key regulatory bottleneck, we had previously proposed that genes that are up-regulated post induction could be part of the signaling pathways which activate the CSR. Knocking out some of these genes which were non-essential and belonged to the bottom of the E. coli regulatory network had provided higher expression of GFP and L-asparaginase.ResultsWe chose the best performing double knockout E. coli BW25113ΔelaAΔcysW and demonstrated its ability to enhance the expression of the toxic Rubella E1 glycoprotein by 2.5-fold by tagging it with sfGFP at the C-terminal end to better quantify expression levels. Transcriptomic analysis of this hyper-expressing mutant showed that a significantly lower proportion of genes got down-regulated post induction, which included genes for transcription, translation, protein folding and sorting, ribosome biogenesis, carbon metabolism, amino acid and ATP synthesis. This down-regulation which is a typical feature of the CSR was clearly blocked in the double knockout strain leading to its enhanced expression capability. Finally, we supplemented the expression of substrate uptake genes glpK and glpD whose down-regulation was not prevented in the double knockout, thus ameliorating almost all the negative effects of the CSR and obtained a further doubling in recombinant protein yields.ConclusionThe study validated the hypothesis that these up-regulated genes act as signaling messengers which activate the CSR and thus, despite having no casual connection with recombinant protein synthesis, can improve cellular health and protein expression capabilities. Combining gene knockouts with supplementing the expression of key down-regulated genes can counter the harmful effects of CSR and help in the design of a truly superior host platform for recombinant protein expression.

Chaperone-Aided in Vitro Renaturation of an Engineered E1 Envelope Protein for Detection of Anti-Rubella Virus IgG Antibodies

The envelope glycoproteins of Rubella virus, E1 and E2, mediate cell tropism, and E1 in particular plays a pivotal role in the fusion of the virus with the endosomal membrane. Both are the prime targets of the humoral immune response. Recombinant variants of the E1 ectodomain as well as E1 antigen preparations from virus lysates are commonly used to detect anti-Rubella immunoglobulins in human sera. Hitherto, recombinant E1 for diagnostic applications has been produced chiefly in eukaryotic expression systems. Here, we report the high-yield overproduction of an engineered E1 ectodomain in the Escherichia coli cytosol and its simple and convenient renaturation into a highly soluble and immunoreactive conformation. C-Terminal fusion to one or two units of the E. coli chaperone SlyD enhances expression, facilitates in vitro refolding, and improves the overall solubility of Rubella E1. As part of this fusion protein, the E1 ectodomain fragment of residues 201-432 adopts an immunoreactive fold, providing a promising tool for the sensitive and specific detection of anti-E1 IgG in Rubella serology. Two disulfide bonds in the membrane-adjacent part of the E1 ectodomain are sufficient to generate conformations with a high and specific antigenicity. The covalently attached chaperone modules do not impair antibody recognition and binding of Rubella E1 when assessed in a heterogeneous immunoassay. SlyD and related folding helpers are apparently generic tools for the expression and refolding of otherwise unavailable proteins of diagnostic or medical importance.

Point mutation of a rubella virus E1 protein T-cell epitope by substitution of single amino acid reversed the restrictive HLA-DR polymorphism: a possible mechanism maintaining HLA polymorphism.

The influence of single amino acid substitutions within a rubella E1 protein T-cell epitope, E1(273-284) on T-cell recognition was studied. Substitutions of an uncharged amino acid A for an E or for a T and substitution of a T for S were found to not significantly reduce the T-cell responses. However, substitution of a charged residue such as E for hydrophobic residues (I, V, or W); D for Q; or a relatively larger size amino acid for polar residues completely abolished the cytotoxicities mediated by E1(273-284)-specific T-cell clone. A set of single amino acid-substituted peptide analogs of E1(273-284) not eliciting cytotoxicity of the T-cell clone was used to test the influence of point mutation of the epitope on HLA DR restrictions. A panel of B-cell lines with different DR4 subtypes was used as targets in cytotoxicity assays to determine the restrictive HLA molecules. Results showed that modification of the T-cell epitope by point mutation could reverse the HLA DR restriction from one allele to other alleles. A model based on these results has been proposed to explain the mechanism balancing major histocompatibility complex (MHC) polymorphism in outbred populations.

Sequence variation and biological activity of rubella virus isolates.

Haemagglutination (HA) by rubella virus is mediated by the E1 glycoprotein. Rubella isolates which haemagglutinate with different avidity have been characterised. A significant reduction of HA titre at pH 6.0 was observed in one isolate in which isoleucine is substituted for threonine at rubella E1 residue 280. This residue is located in an epitope (EP1) which we have previously identified and shown to bind HA inhibiting (HA1) monoclonal antibodies. The isolates studied are also distinguishable by plaque size but no sequence variations in the immunogenic region of E1 were identified which might account for this difference. No correlation was observed between infectivity and binding affinity of neutralising monoclonal antibodies for different rubella virus strains.

Recombinant rubella E1 fusion proteins for antibody screening and diagnosis

Background: Until rubella is eradicated there will be a continuing need for rubella antibody surveillance. Antigen production using recombinant DNA technology may be a viable alternative to traditional techniques of producing antigens for enzyme immunoassays (EIAs). Objectives: To investigate the potential of bacterial fusion proteins containing rubella E1 protein sequences for use in EIAs to detect rubella antibodies. Study design: Purified bacterial fusion proteins containing rubella E1 sequences to be used as antigens in EIAs and compared to ‘traditional’ assays using virus derived antigens for rubella antibody screening. Results: cDNA clones coding for the complete rubella E1 protein sequence and subfragments of E1 were modified for expression as carboxy terminal fusions with either β-galactosidase or glutathione-S-transferase. β-galactosidase fusions with the complete E1 coding sequence or amino acids 201 to 307, which contain known epitopes, resulted in the production of predominantly insoluble fusion proteins unsuitable for use in EIA. Nine glutathione-S-transferase-E1 fusion proteins were produced with individual fusion proteins exhibiting varying properties with regard to the levels of protein produced, apparent stability, solubility and the potential for affinity purification using glutathione agarose. Reduction of the E1 component to only 44 amino acids containing three B-cell epitopes (Terry et al., 1988) produced an abundant soluble GST-E1 fusion protein (3.5 μg/ml), which could be affinity purified using glutathione agarose. This fusion protein has been successfully used in EIA to detect rubella antibodies. Conclusions: We have shown that GST-E1 fusions have potential as antigens in tests for rubella antibodies.

Structure and antigenic activity of rubella E1 glycoprotein synthetic peptides

Minimal sequences of rubella E1 glycoprotein epitopes were previously identified as the tripeptide 250PER252 for the EP2 epitope, the tetrapeptide 260ADDP263 for the EP3 epitope, and the tripeptide 273EVW275 plus the octapeptide 278PVIGSQAR285 for the EP1 epitope. In order to establish for each epitope the shortest sequence that was able to give the maximum binding with human antirubella immunoglobulins, synthetic peptides with increasing number of residues flanking these essential parts of rubella E1 glycoprotein epitopes were synthesized and examined for their antigenic activity. Usually higher activity was observed with progressively longer homologues, whereas the additions of Pro-271, Pro-278 to 272GEVWVT277 peptide, and additions of Ala-248 to 249TPERP253 and 249TPERPR254, led to an abrupt decrease in binding. Taken together, our results indicated that the antigenic activity of the whole antigen could be dissected and reproduced using synthetic peptides of appropriate structure for each epitope.

Structure of rubella E1 glycoprotein epitopes established by multiple peptide synthesis

Essential parts of epitopes have been identified on rubella virion envelope glycoprotein E1, by scanning with overlapping octapeptides situated between amino acids 243-286 in a previously determined antigenic domain.

A bio-engineered rubella E1 antigen.

The major rubella envelope protein, E1, and a segment of it, comprising amino acids 207-353, have been separately expressed as fusion proteins with the IgG binding region of Staphylococcus aureus protein A in Escherichia coli. The proteins carry E1-specific antigenicity recognized by monoclonal antibodies raised against whole virus confirming that correct glycosylation is not required for antigenicity. The use of these bioengineered antigens in immunoassays for diagnosis of rubella infection and for immunization in experimental animals is described.