Cloning, Expression, and Purification of Histidine-Tagged preS Domains of Hepatitis B Virus

Núñez, Elena; Wei, Xin; Delgado, Carmen; Rodrı́guez-Crespo, Ignacio; Yelamos, Belèn; Gómez-Gutiérrez, Julián; Gavilanes, Francisco

doi:10.1006/prep.2000.1368

Cited by 17 publications

(6 citation statements)

References 32 publications

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“…The increment in positive charge as the pH is diminished would favor the initial step and hence the insertion into the bilayer. It is worth noting that these differences cannot be attributed to a conformational change of the protein but rather to a modification in their ionic state since the spectroscopic properties at pH 5.0 are coincident with those previously described at pH 7.0 [16].…”

Section: Discussionsupporting

confidence: 78%

“…The cDNAs coding for preS domains of subtypes adw and ayw were cloned as described previously in expression vectors that add six-histidine sequences at the carboxy-terminal end of each protein [16]. Escherichia coli strains BL21 (DE3) and HMS174 (DE3) were transformed with the recombinant plasmids pT7-7-preS-his-adw and pET-3d-preS-his-ayw respectively and isopropyl -Dthiogalactopiranosyde (IPTG) was added to a final concentration of 0.5 mM to induce protein expression.…”

Section: Cloning Expression Purification and Labeling Of Pres Domainsmentioning

confidence: 99%

“…Escherichia coli strains BL21 (DE3) and HMS174 (DE3) were transformed with the recombinant plasmids pT7-7-preS-his-adw and pET-3d-preS-his-ayw respectively and isopropyl -Dthiogalactopiranosyde (IPTG) was added to a final concentration of 0.5 mM to induce protein expression. Both recombinant proteins were purified using a single affinity-chromatography step in Sepharose CL-6B Ni-nitrilotriacetic acid (NTA) column (Qiagen), following procedures previously described, giving rise to highly pure and stable 20-25 mg of preS-his-ayw and 35-40 mg of preS-his-adw from 1L of culture media [16].…”

Section: Cloning Expression Purification and Labeling Of Pres Domainsmentioning

confidence: 99%

“…Despite their location at the surface of natural virions, the preS domains have never been directly involved in the fusion process, being their role in viral entry only associated with the attachment of HBV particles to possible cellular receptors. With the aim to explore the fusogenic capabilities of these regions, two recombinant preS domains from adw and ayw subtypes, produced in E. Coli cells 5 as previously described [16] were used in membrane interaction studies. In this paper we describe that preS domains are able to interact with acidic phospholipid vesicles and to destabilize these membrane model systems, and hence, could contribute, together with the N-terminal S peptide, to the fusion of viral and cellular membranes.…”

Section: Introductionmentioning

confidence: 99%

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Interaction of preS domains of hepatitis B virus with phospholipid vesicles

Núñez

Yelamos

Delgado

et al. 2009

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The role of preS domains of the hepatitis B virus (HBV) envelope proteins in the first steps of viral infection has been restricted to their implication in virus attachment to a putative hepatocyte receptor. In order to explore a fusion activity in these regions, we used recombinant preS domains to characterize their interaction with liposomes. Binding experiments carried out with NBD-labeled proteins indicated that preS were able to interact in a monomeric way with acidic phospholipid vesicles, being the partition coefficient similar to that described for peptides which can insert deeply into bilayers. Fluorescence depolarization of DPH-labeled vesicles confirmed the specificity for negative charged phospholipids. Upon interaction the proteins induced aggregation, lipid mixing and release of internal contents of acidic vesicles at both acid and neutral pH in a concentration-dependent manner. Taken together, all these data indicate that preS domains are able to insert into the hydrophobic core of the bilayer. Moreover, the insertion resulted in a protein conformational change which increased the helical content. Therefore all these results suggest that, besides their participation in the recognition of a cellular receptor, the preS domains could be involved in the fusion mechanism of HBV with the plasma membrane of target cells.

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Section: Discussionsupporting

confidence: 78%

Section: Cloning Expression Purification and Labeling Of Pres Domainsmentioning

confidence: 99%

Section: Cloning Expression Purification and Labeling Of Pres Domainsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Interaction of preS domains of hepatitis B virus with phospholipid vesicles

Núñez

Yelamos

Delgado

et al. 2009

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…Overexpression of the preS‐related proteins had produced insoluble proteins that were accumulated in inclusion bodies (Lin et al. 1991; Núñez et al. 2001).…”

Section: Discussionmentioning

confidence: 99%

Delivery of chimeric hepatitis B core particles into liver cells

Lee¹,

Tey²,

Ho³

et al. 2011

Journal of Applied Microbiology

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Aims: To display a liver‐specific ligand on the hepatitis B virus core particles for cell‐targeting delivery. Methods and Results: A liver cell–binding ligand (preS1) was fused at the N‐terminal end of the hepatitis B core antigen (HBcAg), but the fusion protein (preS1His6HBcAg) was insoluble in Escherichia coli and did not form virus‐like particles (VLPs). A method to display the preS1 on the HBcAg particle was established by incorporating an appropriate molar ratio of the truncated HBcAg (tHBcAg) to the preS1His6HBcAg. Gold immunomicroscopy showed that the subunit mixture reassembled into icosahedral particles, displaying the preS1 ligand on the surface of VLPs. Fluorescence microscopy revealed that the preS1 ligand delivered the fluorescein‐labelled VLPs into the HepG2 cells efficiently. Conclusions: Chimeric VLPs containing the insoluble preS1His6HBcAg and highly soluble tHBcAg were produced by a novel incorporation method. The preS1 ligand was exposed on the surface of the VLPs and was shown to deliver fluorescein molecules into liver cells. Significance and Impact of Study: The newly established incorporation method can be used in the development of chimeric VLPs that could serve as potential nanovehicles to target various cells specifically by substituting the preS1 ligand with different cell‐specific ligands.

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Programmed hepatocytes cell death associated with FLIP downregulation in response to extracellular PreS1/2

Rojas

Barboza

Terán-Ángel

et al. 2013

Journal of Medical Virology

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Chronic hepatitis B virus (HBV) infection involves liver damage resulting in continuous cell injury and death. During HBV infection, hepatocytes exhibit changes in death receptor expression and in their susceptibility to death. These changes are observed not only in infected cells but also in bystander cells. Because excess viral surface protein (HBsAg) is secreted in large amounts as soluble particles containing preS proteins, the role of soluble preS1/2 in hepatocyte (HepG2) death modulation is an important issue to be explored. An increase of cell death induced by preS1/2 was observed. Also, cell death was associated with the down-regulation of FLIP and activation of caspase 8, caspase 9, and BID. Additionally, hepatocytes exhibited a sensitization to death mediated by the Fas receptor. These results, may contribute to understanding the role of envelope proteins (preS1/2) in the pathogenesis of HBV infection.

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Cloning, Expression, and Purification of Histidine-Tagged preS Domains of Hepatitis B Virus

Cited by 17 publications

References 32 publications

Interaction of preS domains of hepatitis B virus with phospholipid vesicles

Interaction of preS domains of hepatitis B virus with phospholipid vesicles

Delivery of chimeric hepatitis B core particles into liver cells

Programmed hepatocytes cell death associated with FLIP downregulation in response to extracellular PreS1/2

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