Dataset Information

In vitro-Formulated Oligomers of Strep-Tagged Avian Influenza Haemagglutinin Produced in Plants Cause Neutralizing Immune Responses.

ABSTRACT: The worldwide emergence of the novel influenza A H5N1 and H5N8 has notably and directly impacted the poultry industry, resulting in the need for effective and cheap vaccination strategies to protect poultry worldwide. Subunit vaccines from plants can be produced for a low cost, and plant production systems are easily scaled up at low infrastructure cost. However, subunit vaccines generally induce low immunogenicity against influenza. To address this issue, we present a new and innovative method to generate highly immunogenic H5 oligomers. The method is based on specific and high-affinity interaction between engineered streptavidin (Strep-Tactin® XT) and the Strep-tag II peptide. H5-Strep-tag II-tagged trimers were produced via transient agroinfection in tobacco leaves and purified, and oligomers were formulated in vitro by adding purified homotetrameric Strep-Tactin® XT. Immunogenicity was tested by performing mouse immunizations. Haemagglutinin oligomers produced in vitro by combining Strep-Tactin® XT and Strep-tag II-fused haemagglutinin trimers from plants raised potentially neutralizing antibodies in mice. Vaccines based on actual H5N1 haemagglutinin can be produced by combining strep-tagged haemagglutinin trimers from plants and Strep-Tactin® XT.

SUBMITTER: Phan HT

PROVIDER: S-EPMC6110258 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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<i>In vitro</i>-Formulated Oligomers of Strep-Tagged Avian Influenza Haemagglutinin Produced in Plants Cause Neutralizing Immune Responses.

Phan Hoang Trong HT Gresch Ulrike U Conrad Udo U

Frontiers in bioengineering and biotechnology 20180820

The worldwide emergence of the novel influenza A H5N1 and H5N8 has notably and directly impacted the poultry industry, resulting in the need for effective and cheap vaccination strategies to protect poultry worldwide. Subunit vaccines from plants can be produced for a low cost, and plant production systems are easily scaled up at low infrastructure cost. However, subunit vaccines generally induce low immunogenicity against influenza. To address this issue, we present a new and innovative method ...[more]

PMID: 30177967

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Project description:Human cannabinoid receptor CB2 belongs to the class A of G protein-coupled receptor (GPCR). CB2 is predominantly expressed in membranes of cells of immune origin and is implicated in regulation of metabolic pathways of inflammation, neurodegenerative disorders and pain sensing. High resolution structural studies of CB2 require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recombinant CB2 and its stabilization in a functional state, here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB2 with the resin, the double repeat of the Strep-tag (a sequence of eight amino acids WSHPQFEK), named the Twin-Strep-tag was attached either to the N- or C-terminus of CB2 via a short linker, and the recombinant protein was expressed in cytoplasmic membranes of E. coli as a fusion with the N-terminal maltose binding protein (MBP). The CB2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed on the purified protein demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The binding capacity of the resin was several-fold higher for the tag located at the N-terminus of the protein as opposed to the C-terminus- or middle of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies.

Dataset Information

In vitro-Formulated Oligomers of Strep-Tagged Avian Influenza Haemagglutinin Produced in Plants Cause Neutralizing Immune Responses.

Publications

<i>In vitro</i>-Formulated Oligomers of Strep-Tagged Avian Influenza Haemagglutinin Produced in Plants Cause Neutralizing Immune Responses.

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