Abstract
Tight junctions (TJs) are the outermost structures of intercellular junctions and are highly specialized membrane domains involved in many important cellular processes. However, most TJ proteins are four-time transmembrane proteins and are difficult to express in their correct soluble form, which limits their functional study and therapeutic application. Human occludin (OCLN) is a major component of TJs and an essential co-receptor for hepatitis C virus (HCV) cell entry. To explore expression strategy for recombinant TJ proteins possessing integrated and functional extracellular loops, OCLN was here used as a model molecule, and several prokaryotic fusion constructs were designed by docking OCLN extracellular loops (ECLs) to HIV-1 gp41 NHR and CHR six-helical bundle (6HV1); then their biophysical features and anti-HCV activity were evaluated. The proteins were successfully expressed and purified in E. coli, and the double-loop constructs (D1ECL1S+D2ECL2 as a representative) were found to have more potent HCV neutralizing activity than single-loop constructs at non-cytotoxic concentrations. Circular dichroism studies indicate that D1ECL1S+D2ECL2 adopt stable α-helical folds consistent with design. Thermal denaturation assay indicated that D1ECL1S+D2ECL2 is highly stable at 80°C (melting temperature, Tm, of 89.08 ± 2.0°C) and comparable in stability to the 6HV1 scaffold. Moreover, the time-of-addition experiment revealed that D1ECL1S+D2ECL2 predominantly functioned during the early stages of HCV entry. Taken together, these findings provide a novel strategy for recombinant TJ protein expression in vitro, which may shed light on functional and structural studies for TJs and may provide a new avenue to drug development.
Highlights
Tight junctions (TJs) are present at the apical ends of lateral membrane surfaces of epithelial and endothelial cells and form a series of discrete sites of apparent membrane fusion involving the outer leaflet of plasma membranes of adjacent cells
Linkers were used to string together the N36 and C34 (Fig 1B). This tandem protein was used to simulate the transmembrane domains of OCLN to ensure correct conformation and the domain 1 (D1) and domain 2 (D2) can be replaced by other protein domains without influence the structure
6HV1 D1 and D2 were replaced with OCLN ECL1S, ECL1L, or ECL2 (Table 1)
Summary
Tight junctions (TJs) are present at the apical ends of lateral membrane surfaces of epithelial and endothelial cells and form a series of discrete sites of apparent membrane fusion involving the outer leaflet of plasma membranes of adjacent cells. Fusion expression Occludin extracellular loops and α-helical bundle proteins involved in TJs, including transmembrane proteins such as occludin (OCLN), claudins, junctional adhesion molecules, and cytoplasmic adaptor molecules such as ZO-1, ZO-2 and ZO-3[1]. TJs play key roles in the barrier function and fence function in normal cells. The close relationship between TJs and diseases is widely recognized, lack of efficient expression method of recombinant TJ proteins hindered intensive structural and functional studies for TJs. the close relationship between TJs and diseases is widely recognized, lack of efficient expression method of recombinant TJ proteins hindered intensive structural and functional studies for TJs This is an obstacle for targeting TJs during drug development
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