Efficient Mammalian Expression Research Articles

Immunogenicity and protective efficacy of a trimeric full-length S protein subunit vaccine for porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) has caused serious economic losses to the pig husbandry worldwide, and the effects of existing commercialized vaccines are suboptimal. Therefore, research to develop an efficacious vaccine for prevention and control of PEDV is essential. In this study, we designed and produced trimerized proteins of full-length PEDV spike (S) protein, S1 subunit, and a tandem of multiple epitopes of S protein using an efficient mammalian expression vector system in HEK 293F cells. The immunogenicity of two commercial adjuvants, M401 and M103, was also evaluated in mice. Enzyme-linked immunosorbent assays demonstrated that all immunized mice generated highly systemic PEDV S-specific IgG and IgA antibodies. Mice in S/M103-immunized group generated the highest neutralizing antibody titer with 1:96. Compared with control group, the subunit vaccines elicited multifunctional CD3+CD4+ and CD3+CD8+ T cells, B220+CD19+ B cells, and CD3-CD49b+ natural killer cells in the spleen. PEDV S/M103 vaccine, which had the best immune effect, was selected for further evaluation in piglets. Immunization with S/M103 vaccine induced high levels of S-specific IgG, IgA, and neutralizing antibodies, and increased the proliferation of peripheral blood mononuclear cells and the expression levels of interferon-γ and interleukin-4 in peripheral blood of piglets. Virus challenge test results showed significantly lower diarrheal index scores and fecal viral loads, and less pathological damage to the intestines in S/M103-immunized piglets than in controls, indicating that S/M103 provides good protection against the virulent virus challenge. Our findings suggest that trimeric PEDV S/M103 has potential as a clinical vaccine candidate.

Mammalian expression of virus-like particles as a proof of principle for next generation polio vaccines

Global vaccination programs using live-attenuated oral and inactivated polio vaccine (OPV and IPV) have almost eradicated poliovirus (PV) but these vaccines or their production pose significant risk in a polio-free world. Recombinant PV virus-like particles (VLPs), lacking the viral genome, represent safe next-generation vaccines, however their production requires optimisation. Here we present an efficient mammalian expression strategy producing good yields of wild-type PV VLPs for all three serotypes and a thermostabilised variant for PV3. Whilst the wild-type VLPs were predominantly in the non-native C-antigenic form, the thermostabilised PV3 VLPs adopted the native D-antigenic conformation eliciting neutralising antibody titres equivalent to the current IPV and were indistinguishable from natural empty particles by cryo-electron microscopy with a similar stabilising lipidic pocket-factor in the VP1 β-barrel. This factor may not be available in alternative expression systems, which may require synthetic pocket-binding factors. VLPs equivalent to these mammalian expressed thermostabilized particles, represent safer non-infectious vaccine candidates for the post-eradication era.

Establishment of a mammalian expression system for recombinant [-2]proPSA and a specific antibody against the truncated leader peptide.

A truncated precursor form of prostate-specific antigen (PSA), [-2]proPSA, is a well-known biomarker for prostate cancer. To develop a biomarker assay, highly purified [-2]proPSA is required as a standard reference and for generation of a specific antibody. In this study, we generated an efficient mammalian expression system for producing a recombinant [-2]proPSA-human kappa constant domain (Cκ ) fusion protein. N-terminal amino acid sequencing using Edman degradation demonstrated that over 95% of the recombinant protein produced is [-2]proPSA, thereby showing for the first time that recombinant [-2]proPSA can be produced as a major fraction. We also generated a recombinant chicken antibody specific to [-2]proPSA but not cross-reactive to recombinant [-7]proPSA-Cκ , [-5]proPSA-Cκ , and PSA purified from human seminal fluid in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Also, the recombinant chicken antibody reacted to recombinant [-2]proPSA protein bound to an anti-PSA antibody coated on the micrometer plate in a sandwich ELISA. All of these results suggest that the N-terminus of the [-2]proPSA-Cκ fusion protein resides on the exterior of the protein, thus allowing exposure to the antibody.

Direct Expression and Validation of Phage-selected Peptide Variants in Mammalian Cells

Phage display is a key technology for the identification and maturation of high affinity peptides, antibodies, and other proteins. However, limitations of bacterial expression restrict the range and sensitivity of assays that can be used to evaluate phage-selected variants. To address this problem, selected genes are typically transferred to mammalian expression vectors, a major rate-limiting step in the iterative improvement of peptides and proteins. Here we describe a system that combines phage display and efficient mammalian expression in a single vector, pDQ1. This system permits immediate expression of phage-selected genes as IgG1-Fc fusions in mammalian cells, facilitating the rapid, sensitive characterization of a large number of library outputs for their biochemical and functional properties. We demonstrate the utility of this system by improving the ability of a CD4-mimetic peptide to bind the HIV-1 envelope glycoprotein and neutralize HIV-1 entry. We further improved the potency of the resulting peptide, CD4mim6, by limiting its ability to induce the CD4-bound conformation of the envelope glycoprotein. Thus, CD4mim6 and its variants can be used to investigate the properties of the HIV-1 envelope glycoprotein, and pDQ1 can accelerate the discovery of new peptides and proteins through phage display.

Signal peptide and denaturing temperature are critical factors for efficient mammalian expression and immunoblotting of cannabinoid receptors

Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immuno-blotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immuno-blot analysis. These findings provide very useful information for efficient mammalian expression and immuno-blotting of membrane receptors.

Lassa virus-like particles displaying all major immunological determinants as a vaccine candidate for Lassa hemorrhagic fever.

BackgroundLassa fever is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. Treatment of acute Lassa fever infections has successfully utilized intravenous administration of ribavirin, a nucleotide analogue drug, but this is not an approved use; efficacy of oral administration has not been demonstrated. To date, several potential new vaccine platforms have been explored, but none have progressed toward clinical trials and commercialization. Therefore, the development of a robust vaccine platform that could be generated in sufficient quantities and at a low cost per dose could herald a subcontinent-wide vaccination program. This would move Lassa endemic areas toward the control and reduction of major outbreaks and endemic infections. To this end, we have employed efficient mammalian expression systems to generate a Lassa virus (LASV)-like particle (VLP)-based modular vaccine platform.ResultsA mammalian expression system that generated large quantities of LASV VLP in human cells at small scale settings was developed. These VLP contained the major immunological determinants of the virus: glycoprotein complex, nucleoprotein, and Z matrix protein, with known post-translational modifications. The viral proteins packaged into LASV VLP were characterized, including glycosylation profiles of glycoprotein subunits GP1 and GP2, and structural compartmentalization of each polypeptide. The host cell protein component of LASV VLP was also partially analyzed, namely glycoprotein incorporation, though the identity of these proteins remain unknown. All combinations of LASV Z, GPC, and NP proteins that generated VLP did not incorporate host cell ribosomes, a known component of native arenaviral particles, despite detection of small RNA species packaged into pseudoparticles. Although VLP did not contain the same host cell components as the native virion, electron microscopy analysis demonstrated that LASV VLP appeared structurally similar to native virions, with pleiomorphic distribution in size and shape. LASV VLP that displayed GPC or GPC+NP were immunogenic in mice, and generated a significant IgG response to individual viral proteins over the course of three immunizations, in the absence of adjuvants. Furthermore, sera from convalescent Lassa fever patients recognized VLP in ELISA format, thus affirming the presence of native epitopes displayed by the recombinant pseudoparticles.ConclusionsThese results established that modular LASV VLP can be generated displaying high levels of immunogenic viral proteins, and that small laboratory scale mammalian expression systems are capable of producing multi-milligram quantities of pseudoparticles. These VLP are structurally and morphologically similar to native LASV virions, but lack replicative functions, and thus can be safely generated in low biosafety level settings. LASV VLP were immunogenic in mice in the absence of adjuvants, with mature IgG responses developing within a few weeks after the first immunization. These studies highlight the relevance of a VLP platform for designing an optimal vaccine candidate against Lassa hemorrhagic fever, and warrant further investigation in lethal challenge animal models to establish their protective potential.

High efficient mammalian expression and secretion of a functional humanized single-chain Fv/human interleukin-2 molecules

To construct and produce a recombinant bispecific humanized single-chain Fv (sFv) /Interleukin-2 (IL-2) fusion protein by using mammalian cells. The sFv/IL-2 protein was genetically engineered, and transfected to mammalian cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv/IL-2 with high efficiency. The fusion protein was constructed and high efficiently expressed with yields up to 102 +/- 4.2 mg/L in culture supernatant of the stably transfected 293 cell line. This recombinant fusion protein consisted of humanized variable heavy (V(H)) and light (V(L)) domains of monoclonal antibody (mAb) 520C9 directed against the human HER-2/neu (c-erbB2) proto-oncogene product p185, and human IL-2 connected by polypeptide linker. The fusion protein was shown to retain the immunostimulatory activities of IL-2 as measured by IL-2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activities, this fusion protein also possessed antigen-binding specificity against p185, as determined by indirect ELISA using p185 positive SKOV 3ip1 cells. The large-scale preparation of the recombinant humanized sFv antibody/IL-2 fusion protein is performed with 293 cells. The recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means of targeting therapeutic doses of IL-2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.

The human elongation factor 1 alpha (EF-1α) first intron highly enhances expression of foreign genes from the murine cytomegalovirus promoter

In order to develop a highly efficient mammalian expression vector, we constructed a vector by the combination of the murine cytomegalovirus (MCMV) immediate early (IE) promoter and the human elongation factor one alpha (EF-1α) first intron. The MCMV IE promoter was several fold stronger than the human cytomegalovirus (HCMV) immediate early (IE) promoter and the human elongation factor one alpha (EF-1α) promoter in various mammalian cell lines such as NIH3T3, Neuro-2a, 293T or HT1080 and was only slightly weaker than the HCMV or the EF-1α promoter in HeLa and CHO cell lines. We inserted the first intron of the human EF-1α gene behind the MCMV and the HCMV promoter to enhance the gene expression through increasing RNA transcription and/or RNA stability. The insertion of the human EF-1α first intron markedly enhanced the level of gene expression in many cell lines and the resultant MCMV promoter with the human EF-1α first intron (MCMV/EF-I) was higher than the HCMV promoter from 4.3 to 65.5-fold in various cell lines. Also, the MCMV/EF-I promoter induced higher level of erythropoietin expression than the HCMV promoter in transiently transfected CHO cells.

The isolation and identification of cDNA genes by their heterologous expression and function.

Functional cDNA genes for cell surface proteins, receptors, growth factors and nuclear and cytoplasmic proteins can be isolated by a heterologous expression-function gene isolation strategy. The method of this strategy is dependent on highly efficient mammalian expression vectors, the properties of COS cells as hosts for SV40-origin-containing mammalian expression vectors, the well-established technology of constructing highly complex plasmid cDNA libraries and on the sensitivity of biological assays. These technologies are combined to identify a specific gene sequence by its biological phenotype. In theory this can be accomplished by individually testing each single gene of the cDNA library by expression in COS cells. In practice complex cDNA libraries are formed so that multiple cDNA genes can be tested and analyzed simultaneously. When the biological assay can discriminate phenotypic changes at the single cell level as typified by cellular morphology, cell membrane proteins or receptors or intracellular enzymes, then a reiterative expression screening method can be used. This was demonstrated by the isolation of cDNA genes encoding cell membrane proteins (7-10, 12). For the isolation of cDNA genes for growth factors and cytoplasmic enzymes by the expression-function cDNA gene isolation strategy, an alternative method is followed. The cDNA library is partitioned into sets of cDNA clones for transfection and analysis. The number of clones in a set is dependent on the efficiency of the expression vector used and on the sensitivity of the biological assay. For secreted cytokines and growth factors which can be simply detected by factor dependent cell lines, about 1,000 cDNA clones can be tested as a single set. By this strategy the genes for numerous hematopoietic growth factors, cytokines and interleukins have been isolated. With such numerous successes we can expect that the heterologous expression-function cDNA gene isolation strategy will become an important molecular biology method.

Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase.

DNA coding for bacteriophage T7 RNA polymerase was ligated to a vaccinia virus transcriptional promoter and integrated within the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and stably expressed T7 RNA polymerase in mammalian cells. Target genes were constructed by inserting DNA segments that code for beta-galactosidase or chloramphenicol acetyltransferase into a plasmid with bacteriophage T7 promoter and terminator regions. When cells were infected with the recombinant vaccinia virus and transfected with plasmids containing the target genes, the latter were expressed at high levels. Chloramphenicol acetyltransferase activity was 400-600 times greater than that observed with conventional mammalian transient-expression systems regulated either by the enhancer and promoter regions of the Rous sarcoma virus long terminal repeat or by the simian virus 40 early region. The vaccinia/T7 hybrid virus forms the basis of a simple, rapid, widely applicable, and efficient mammalian expression system.