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

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.紧密连接（Tight junctions, TJs）是细胞间连接的最外层结构，属于高度特化的膜结构域，参与诸多重要细胞生理过程。然而，绝大多数紧密连接蛋白为四次跨膜蛋白，难以获得正确可溶的表达形式，这极大限制了其功能研究与治疗应用。人闭合蛋白（Human occludin, OCLN）是紧密连接的核心组分之一，同时也是丙型肝炎病毒（hepatitis C virus, HCV）侵染细胞所必需的共受体。为探索能够表达具有完整功能性胞外环的重组紧密连接蛋白的策略，本研究以OCLN作为模型分子，通过将OCLN的胞外环（extracellular loops, ECLs）与人类免疫缺陷病毒1型（HIV-1）gp41的N端七肽重复区（NHR）和C端七肽重复区（CHR）的六螺旋束（6HV1）融合，设计了多种原核融合表达构建体，并对其生物物理特性与抗HCV活性进行了评估。上述蛋白均在大肠杆菌（E. coli）中成功表达并纯化，其中以双环构建体D1ECL1S+D2ECL2为代表，在非细胞毒性浓度下展现出比单环构建体更强的HCV中和活性。圆二色光谱分析结果显示，D1ECL1S+D2ECL2呈现出与设计相符的稳定α螺旋折叠结构。热变性实验表明，D1ECL1S+D2ECL2在80℃下仍具备极高稳定性，其解链温度（melting temperature, Tm）为89.08±2.0℃，稳定性与6HV1骨架相当。此外，添加时间实验结果证实，D1ECL1S+D2ECL2主要在HCV侵染的早期阶段发挥作用。综上，本研究为体外重组表达紧密连接蛋白提供了一种全新策略，有望为紧密连接的功能与结构研究提供新思路，并为相关药物开发开辟新路径。