Gene cloning, bacterial expression, in vitro refolding, and characterization of a single-chain Fv antibody against PreS1(21–47) fragment of HBsAg

Yang, Xin-Xiu; Hu, Weiguo; Feng, Liang; Xia, Hengchuan; Zhang, Zuchuan

doi:10.1016/j.pep.2005.02.005

Cited by 21 publications

(15 citation statements)

References 22 publications

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“…Bound MBP-PreS fusion proteins were resolved by SDS-PAGE and analyzed by Western blotting. An anti-PreS1 monoclonal antibody (MAb), 125E11 (21,47), and an anti-MBP MAb (Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies in Western blotting. Signals were visualized using the enhanced chemiluminescence method with a horseradish peroxidase-labeled rabbit anti-mouse immunoglobulin (Dako, Carpenteria, CA).…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of Peptides That Interact with Hepatitis B Virus via the Putative Receptor Binding Site

Deng

Zhai

Michel

et al. 2007

J Virol

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A direct involvement of the PreS domain of the hepatitis B virus (HBV) large envelope protein, and in particular amino acid residues 21 to 47, in virus attachment to hepatocytes has been suggested by many previous studies. Several PreS-interacting proteins have been identified. However, they share few common sequence motifs, and a bona fide cellular receptor for HBV remains elusive. In this study, we aimed to identify PreS-interacting motifs and to search for novel HBV-interacting proteins and the long-sought receptor. PreS fusion proteins were used as baits to screen a phage display library of random peptides. A group of PreS-binding peptides were obtained. These peptides could bind to amino acids 21 to 47 of PreS1 and shared a linear motif (W 1 T 2 X 3 W 4 W 5 ) sufficient for binding specifically to PreS and viral particles. Several human proteins with such a motif were identified through BLAST search. Analysis of their biochemical and structural properties suggested that lipoprotein lipase (LPL), a key enzyme in lipoprotein metabolism, might interact with PreS and HBV particles. The interaction of HBV with LPL was demonstrated by in vitro binding, virus capture, and cell attachment assays. These findings suggest that LPL may play a role in the initiation of HBV infection. Identification of peptides and protein ligands corresponding to LPL that bind to the HBV envelope will offer new therapeutic strategies against HBV infection.Attachment of virions to human hepatocyte membrane via the interaction of the viral envelope protein with a specific cell surface receptor is considered the initial step of hepatitis B virus (HBV) infection. Besides host-derived phospholipids, the envelope of an HBV virion contains virus-derived small (SHBs), middle (MHBs), and large (LHBs) surface proteins translated from distinct initiation codons while sharing a common carboxyl domain (i.e., SHBs). Consequently, LHBs contains an extra N-terminal PreS domain, which is further divided into the amino-PreS1 and carboxyl-PreS2 domains. MHBs contains the PreS2 domain but lacks the PreS1 domain. LHBs is associated mainly with infectious virions but can also be found in smaller amounts on rod-shaped subviral particles. On the other hand, the spherical subviral particles are composed mainly of SHBs. LHBs plays a pivotal role in the infection and budding stages of the HBV life cycle. LHBs proteins in the virion envelope exhibit mixed topologies, with their PreS domains protruding either inwardly or outwardly (32,35

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Section: Methodsmentioning

confidence: 99%

Identification and Characterization of Peptides That Interact with Hepatitis B Virus via the Putative Receptor Binding Site

Deng

Zhai

Michel

et al. 2007

J Virol

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“…According to a previous study by Yang et al (2005), a glycine-and serine-rich linker (G4S)3, could be used to link two genes such as the extracellular regions of SLA-2 and β 2 m to produce a combined fragment by SOE-PCR, and this would aid the study of the interaction between SLA-2 and β 2 m. Because glycine is a simple amino acid lacking side chains, its presence does not affect the structure of β-sheets. Serine often occurs in turns and random coils and the distance between glycine and serine is only 3.5 nm.…”

Section: Discussionmentioning

confidence: 99%

Construction of multiple recombinant SLA-I proteins by linking heavy chains and light chains in vitro and analyzing their secondary and 3-dimensional structures

Feng-shan

Bai

Zhang

et al. 2012

Gene

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“…Choi and colleagues prepared and purified ScAb from anti-mycotoxin deoxy nivalenol (DON) proteins, the activity of which was 1/42 of its parent MAb (Choi et al 2004). Recently, Yang et al (2005) obtained ScAb of the anti-human HBsAg PreS1 segment with the correct folding conformation, and its activity was 1/60 of its parent MAb.…”

Section: Discussionmentioning

confidence: 99%

Cloning, purification and bioactivity assay of human CD28 single-chain antibody in Escherichia coli

Zheng¹,

Qiu²,

Chen³

et al. 2009

Cytotechnology

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Engineered single chain antibodies have become a powerful source of immunotherapy against a wide range of diseases. Here, we present the generation of human CD28 single-chain antibody gene (CD28-ScFv), which contained variable fragments of heavy chain and light chain (VH and VL) of the anti-CD28 antibody, and a linking peptide (Gly4Ser)3 inserted in the middle of VH and VL. The fused gene CD28-ScFv was successfully expressed in BL21 (DE3) cells and confirmed by western blotting assay. The molecular weight of CD28-ScFv was 43 kDa and the major fraction was expressed as an insoluble body. By dissolving the insoluble bodies, renaturing in vitro and purifying with a Ni-NTA affinity column, highly purified expression products of CD28-ScFv were obtained. This product could recognize and bind to CD28+ positive T cells. The proliferation capacity of peripheral blood T cells was increased by purified CD28-ScFv. In this study, we improved orthodox renaturing techniques by combining the dilution renaturation with phase gradient dialysis. With this new method, highly purified CD28-ScFv products were developed and biological activity of the products was similar to that of the mouse monoclonal anti-human CD28 antibody.

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Gene cloning, bacterial expression, in vitro refolding, and characterization of a single-chain Fv antibody against PreS1(21–47) fragment of HBsAg

Cited by 21 publications

References 22 publications

Identification and Characterization of Peptides That Interact with Hepatitis B Virus via the Putative Receptor Binding Site

Identification and Characterization of Peptides That Interact with Hepatitis B Virus via the Putative Receptor Binding Site

Construction of multiple recombinant SLA-I proteins by linking heavy chains and light chains in vitro and analyzing their secondary and 3-dimensional structures

Cloning, purification and bioactivity assay of human CD28 single-chain antibody in Escherichia coli

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