In vivo biotinylated recombinant influenza A virus hemagglutinin for use in subtype-specific serodiagnostic assays

Postel, Alexander; Letzel, Tobias; Müller, Frederik; Ehricht, Ralf; Pourquier, Philippe; Dauber, Malte; Grund, Christian; Beer, Martin; Harder, Timm C.

doi:10.1016/j.ab.2010.12.022

Cited by 14 publications

(8 citation statements)

References 28 publications

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“…The biotin moiety bound to the Avi tag can then be detected via (strept)avidin. It is known that the interaction between biotin and streptavidin or avidin is very strong, sensitive, and selective, so (strept)avidin-biotin binding has been widely used in molecular biology research ( 30 – 33 ).…”

Section: Resultsmentioning

confidence: 99%

“…pB-E rns -V5, pB-E rns /TM-V5 ( 18 ), pcDNA3-VSVg (kindly provided by Stefan Finke, FLI), and pDsRed-ER and pDsRed-Golgi (TaKaRa Bio USA, Inc. [formerly Clontech Laboratories, Inc.]) were used, as well as pCR-13 (BVDV CP7 pCI-E1), pCR-16 (BVDV CP7 pCI-E2-AU1), pCR-17 (BVDV CP7 pCI-E1-E2), and pCR-128 (BVDV CP7 pCI-Flag-E1-V5) ( 17 ). pFBD-shortH5-Avi was kindly provided by Timm Harder, FLI, Insel Riems, Germany ( 33 ). p798, a full-length infectious clone for BVDV CP7 pA/BVDV ( 27 ), was also used.…”

Section: Methodsmentioning

confidence: 99%

“…To make sure this C-terminal Avi tag cannot be cleaved off by signal peptidase, in pYM-45 an R-to-A mutation was introduced at position −3 of the E1 sequence. For pYM-50, the BirA sequence was amplified from pFBD-shortH5-Avi ( 33 ). pYM-51 is based on pYM-50, and sequences coding for the signal sequence of E rns and a KDEL sequence were inserted so that they are expressed at the N or C terminus of BirA, respectively, for its ER retention.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of Membrane Topology and Retention Signal of Pestiviral Glycoprotein E1

Radtke

Tews

et al. 2021

J Virol

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Pestiviruses are members of the family Flaviviridae, a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, Erns, E1 and E2. Among them, E1 is the least characterized concerning both biochemical features and function. E1 from bovine viral diarrhea virus (BVDV) strain CP7 was analyzed with regard to its intracellular localization and membrane topology. Here, it is shown that even in the absence of other viral proteins, E1 is not secreted or expressed at the cell surface, but localizes predominantly in the ER. Using engineered chimeric TM domains with sequences from E1 and vesicular stomatitis virus G protein, the E1 ER-retention signal could be narrowed down to six fully conserved polar residues in the middle part of the transmembrane domain of E1. Retention was observed even when several of these polar residues were exchanged for alanine. Mutations with a strong impact on E1 retention prevented recovery of infectious viruses when tested in the viral context. Analysis of the membrane topology of E1 before and after the signal peptide cleavage via a selective permeabilization and an in vivo labelling approach revealed that mature E1 is a typical type I transmembrane protein with a single span transmembrane anchor at its C-terminus whereas it adopts a hairpin-like structure with the C-terminus located in the ER lumen when the pre-cleavage situation is mimicked by blocking the cleavage site between E1 and E2. Importance The shortage of specific antibodies against E1 making detection and further analysis of E1 difficult, resulted in a lack of knowledge on E1 compared to Erns and E2 with regard to biosynthesis, structure and function. It is known that pestiviruses bud intracellularly. Here, we show that E1 contains its own ER retention signal: six fully conserved polar residues in the middle part of the TM domain are shown to be the determinants for ER retention of E1. Moreover, those six polar residues could serve as a functional group that intensely affect the generation of infectious viral particles. In addition, the membrane topology of E1 has been determined. In this context, we also identified dynamic changes in membrane topology of E1 with the carboxy-terminus located on the luminal side of the ER in the pre-cleavage state and relocation of this sequence upon signal peptidase cleavage. Our work provides the first systematic analysis of the pestiviral E1 protein with regard to its biochemical and functional characteristics.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Characterization of Membrane Topology and Retention Signal of Pestiviral Glycoprotein E1

Radtke

Tews

et al. 2021

J Virol

View full text Add to dashboard Cite

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“…Recombinant immunodominant proteins of infectious organisms are routinely employed in ELISA for the detection of disease-specific antibodies in the body fluids of suspected patients [ 5 , 6 , 17 , 31 – 33 ]. However, immobilization of these proteins on the surface of ELISA plates can be variable and inefficient [ 11 ].…”

Section: Resultsmentioning

confidence: 99%

“…Over the last two decades, specific and efficient capture of biotin-tagged proteins on streptavidin surface by virtue of extremely high-affinity interaction has found use in a diverse range of biological applications [ 1 , 2 ] including immunoassays [ 3 – 6 ], phage display-based affinity selection [ 7 , 8 ], and affinity-based purification [ 9 , 10 ]. Conventional approaches for immobilization of proteins involve their passive adsorption on polystyrene surfaces.…”

Section: Introductionmentioning

confidence: 99%

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection

et al. 2018

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The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.

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Proximity labeling approaches to study protein complexes during virus infection

Zapatero-Belinchón¹,

Carriquí-Madroñal

2021

Proteomics Approaches to Unravel Virus - Vertebrate Host Interactions

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In vivo biotinylated recombinant influenza A virus hemagglutinin for use in subtype-specific serodiagnostic assays

Cited by 14 publications

References 28 publications

Characterization of Membrane Topology and Retention Signal of Pestiviral Glycoprotein E1

Characterization of Membrane Topology and Retention Signal of Pestiviral Glycoprotein E1

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection

Proximity labeling approaches to study protein complexes during virus infection

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