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Fusion expression of Occludin extracellular loops and an α-helical bundle: A new research model for tight junction.

Fusion expression of Occludin extracellular loops and an α-helical bundle: A new research model for tight junction.
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Chi X, Zhao X, Wang W, Niu Y, Cheng M, Liu X, Cui S, Yang W,


Chi X, Zhao X, Wang W, Niu Y, Cheng M, Liu X, Cui S, Yang W, (click to view)

Chi X, Zhao X, Wang W, Niu Y, Cheng M, Liu X, Cui S, Yang W,

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PloS one 2017 04 2712(4) e0175516 doi 10.1371/journal.pone.0175516

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.

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