Improved expression of secretory and trimeric proteins in mammalian cells via the introduction of a new trimer motif and a mutant of the tPA signal sequence

Wang, Jiaye; Song, Wenting; Li, Yan; Chen, Wenjiang; Yang, Dan; Zhong, Guocai; Zhou, Haizhou; Ren, C.; Yu, Haotong; Ling, Hong

doi:10.1007/s00253-011-3297-0

Cited by 45 publications

(25 citation statements)

References 59 publications

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“…Various studies have shown that truncation of the HIV Env from gp160 to gp150 increases the levels of expression on the surface of virus particles [10,32]. In addition, the replacement of the native HIV Env leader sequence with other leader sequences has also been shown to increase expression [10,33,34]. The use of the flexible glycine linker sequence is also expected to prevent the shedding of the gp120 portion of the HIV Env trimer from the surface of the VLPs [23], resulting in higher levels of detection of gp120 on the surface of the VLPs.…”

Section: Discussionmentioning

confidence: 99%

Immunogenicity of HIV-1 Vaccines Expressing Chimeric Envelope Glycoproteins on the Surface of Pr55 Gag Virus-Like Particles

et al. 2020

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The HIV-1 envelope glycoprotein (Env) is present on the surface of the virion at a very low density compared to most other enveloped viruses. Substitution of various parts of the stalk domain of Env (gp41) with the corresponding elements from other viral glycoproteins has been shown to increase Env spike density on the cell membrane and surface of virus-like particles (VLPs). In this study, chimeric Env antigens were generated by replacing the transmembrane and cytoplasmic domains of HIV-1 Env with the corresponding regions from the influenza H5 hemagglutinin (HA) (gp140HA2tr) and by replacing the entire gp41 region of Env with the HA2 subunit of HA (gp120HA2). Recombinant DNA and modified vaccinia Ankara (MVA) vaccines expressing HIV-1 subtype C mosaic Gag and gp150 Env or either of the chimeras were generated. Surprisingly, no significant differences were found in the levels of expression of gp150 Env or either of the chimeras on the surface of cells or on Gag VLPs. Differences were, however, observed in the binding of different monoclonal antibodies to the HIV-1 Env. Monoclonal antibodies, which recognized a V1 / V2 quaternary epitope at the tip of the native Env trimer, bound gp150 and gp140HA2tr chimera but failed to bind to the gp120HA2 chimera. Autologous Tier 2 neutralizing antibodies (NAbs) were produced by rabbits inoculated with DNA and MVA vaccines expressing the gp140HA2tr chimera or gp150 Env, but not those immunized with the gp120HA2 Env. These results showed that the addition of an HA2 stalk to HIV-1 gp120 did not improve immunogenicity, but rather that the full-length gp150 was required for optimal presentation of epitopes for the elicitation of a neutralizing antibody response to HIV-1.

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Section: Discussionmentioning

confidence: 99%

Immunogenicity of HIV-1 Vaccines Expressing Chimeric Envelope Glycoproteins on the Surface of Pr55 Gag Virus-Like Particles

et al. 2020

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“…Recombinant Py S4/CelTOS and TRAP was produced in suspension HEK293F cells cultures, as previously described (Harupa et al, ; Kaushansky et al, ). Briefly, an expression construct containing a tissue plasminogen activator signal (Wang et al, ), the extracellular domain of Py S4/CelTOS (PlasmoDB:PYYM_1436300, amino acids 25–185) or TRAP (PlasmoDB:PYYM_1351500, residues 23–752), and a C‐terminal His tag followed by an AviTag (Beckett, Kovaleva, & Schatz, ) cloned into the pcDNA‐3.4 vector backbone (Thermo Fisher, Waltham, MA, USA) was introduced into HEK293F cells by high‐density transfection with PEI MAX 40000 (Polysciences, Inc., Warrington, PA, USA; Backliwal, Hildinger, Hasija, & Wurm, ; Carbonetti, Oliver, Glenn, Stamatatos, & Sather, ). Cell cultures were then grown for 5 days before purification of the protein from culture supernatants by Ni‐affinity chromatography followed by size exclusion chromatography.…”

Section: Methodsmentioning

confidence: 99%

Plasmodium yoelii S4/CelTOS is important for sporozoite gliding motility and cell traversal

Steel

Pei

Camargo

et al. 2018

Cellular Microbiology

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Gliding motility and cell traversal by the Plasmodium ookinete and sporozoite invasive stages allow penetration of cellular barriers to establish infection of the mosquito vector and mammalian host, respectively. Motility and traversal are not observed in red cell infectious merozoites, and we have previously classified genes that are expressed in sporozoites but not merozoites (S genes) in order to identify proteins involved in these processes. The S4 gene has been described as criticaly involved in Cell Traversal for Ookinetes and Sporozoites (CelTOS), yet knockout parasites (s4/celtos¯) do not generate robust salivary gland sporozoite numbers, precluding a thorough analysis of S4/CelTOS function during host infection. We show here that a failure of oocysts to develop or survive in the midgut contributes to the poor mosquito infection by Plasmodium yoelii (Py) s4/celtos¯ rodent malaria parasites. We rescued this phenotype by expressing S4/CelTOS under the ookinete-specific circumsporozoite protein and thrombospondin-related anonymous protein-related protein (CTRP) promoter (S4/CelTOS ), generating robust numbers of salivary gland sporozoites lacking S4/CelTOS that were suitable for phenotypic analysis. Py S4/CelTOS sporozoites showed reduced infectivity in BALB/c mice when compared to wild-type sporozoites, although they appeared more infectious than sporozoites deficient in the related traversal protein PLP1/SPECT2 (Py plp1/spect2¯). Using in vitro assays, we substantiate the role of S4/CelTOS in sporozoite cell traversal, but also uncover a previously unappreciated role for this protein for sporozoite gliding motility.

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“…Altering the signal peptide sequence has previously been shown to increase secretion for some proteins [40, 41]. We exchanged the natural signal peptide of sCD4 with three commonly used signal peptides and analyzed the resulting amino acid sequences with the bioinformatics program SignalP, which discriminates secreted from non-secreted proteins [42].…”

Section: Resultsmentioning

confidence: 99%

Lentiviral expression system for the purification of secreted proteins from human cell cultures

et al. 2016

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BackgroundRecombinant proteins of therapeutic use are ideally produced in human cells to ensure appropriate co- and post-translational modifications. However, purification of secreted proteins from the culture media is impeded by low expression from transfected cell lines and the presence of serum proteins. Here we describe a simple and cost-effective approach based on lentiviral vector-mediated gene delivery and expression of a secreted His-tagged protein from human embryonic kidney 293 T cells and direct affinity chromatography purification from the cell culture media.ResultsUsing a protein-based HIV entry inhibitor, soluble CD4 (sCD4), we demonstrated that 293 T cells transduced with a lentiviral vector mediated over 10-fold higher secretion of sCD4 in comparison to 293 T cells transfected with the corresponding plasmid. Secretion of sCD4 increased with the dose of the lentiviral vector up to a multiplicity of infection of 50. Exchanging the native signal peptide of sCD4 with the signal peptide of human alpha-1 antitrypsin increased expression by 50 %. There was no difference in expression from a monocistronic or bicistronic lentiviral vector. Reduction of the serum concentration in the culture media had no significant effect on the secretion of sCD4. Small-scale purification from 50 ml of culture media with reduced serum content yielded up to 1 mg of pure sCD4. Purified sCD4 bound to recombinant HIV envelope glycoprotein 120 (Env gp120) and inhibited HIV entry at concentrations comparable to published results.ConclusionThe procedure outlined in this study can be performed without the need for specialized reagents or equipment and could easily be adapted by any laboratory. Furthermore, the method could be used to produce sCD4 fusion proteins or other His-tagged proteins.

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Improved expression of secretory and trimeric proteins in mammalian cells via the introduction of a new trimer motif and a mutant of the tPA signal sequence

Cited by 45 publications

References 59 publications

Immunogenicity of HIV-1 Vaccines Expressing Chimeric Envelope Glycoproteins on the Surface of Pr55 Gag Virus-Like Particles

Immunogenicity of HIV-1 Vaccines Expressing Chimeric Envelope Glycoproteins on the Surface of Pr55 Gag Virus-Like Particles

Plasmodium yoelii S4/CelTOS is important for sporozoite gliding motility and cell traversal

Lentiviral expression system for the purification of secreted proteins from human cell cultures

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