人干扰素γ(hIFN-γ)作为可溶性细胞因子在先天和适应性免疫中具有多种功能,起着关键作用。在之前的调查中,我们确定了三个关键的氨基酸残基,即,IFN-γ结构上的苏氨酸(T)27,苯丙氨酸(F)29和亮氨酸(L)30,它们是抗IFN-γ自身抗体识别的表位的组成部分。阻止该表位与自身抗体之间的相互作用对于成人发作性免疫缺陷(AOID)的有效治疗至关重要。然而,挑战来自T27AF29L30A突变体在大肠杆菌BL21(DE3)中的溶解度降低。本研究探讨了旨在提高IFN-γT27AF29AL30A的可溶性表达的靶向策略。这是通过利用5种分子伴侣质粒来实现的:pG-KJE8、pKJE7、pGro7、pG-Tf2和pTf16。这些质粒,编码细胞质伴侣,在大肠杆菌BL21(DE3)中与IFN-γ突变体共表达,我们使用SDS-PAGE和Western印迹分析了细胞裂解物和包涵体中的蛋白质。我们的发现揭示了pG-KJE8的显着功效,它容纳了细胞质伴侣DnaK-DnaJ-GrpE和GroEL-GroES,显着增强IFN-γT27AF29AL30A的溶解度。重要的是,这种共表达不仅解决了溶解度问题,而且保留了功能性二聚结构,如通过夹心ELISA证实的。这个有希望的结果标志着在开发AOID的生物学策略方面迈出了重要的一步。
Human interferon gamma (hIFN-γ) plays a pivotal role as a soluble cytokine with diverse functions in both innate and adaptive immunity. In a previous investigation, we pinpointed three critical amino acid residues, i.e., threonine (T) 27, phenylalanine (F) 29, and leucine (L) 30, on the IFN-γ structure, which are integral to the epitope recognized by anti-IFN-γ autoantibodies. It is crucial to impede the interaction between this epitope and autoantibodies for effective therapy in adult-onset immunodeficiency (AOID). However, the challenge arises from the diminished solubility of the T27AF29L30A mutant in Escherichia coli BL21(DE3). This study delves into a targeted strategy aimed at improving the soluble expression of IFN-γ T27AF29AL30A. This is achieved through the utilization of five chaperone plasmids: pG-KJE8, pKJE7, pGro7, pG-Tf2, and pTf16. These plasmids, encoding cytoplasmic chaperones, are co-expressed with the IFN-γ mutant in E. coli BL21(DE3), and we meticulously analyze the proteins in cell lysate and inclusion bodies using SDS-PAGE and Western blotting. Our findings reveal the remarkable efficacy of pG-KJE8, which houses cytoplasmic chaperones DnaK-DnaJ-GrpE and GroEL-GroES, in significantly enhancing the solubility of IFN-γ T27AF29AL30A. Importantly, this co-expression not only addresses solubility concerns but also preserves the functional dimerized structure, as confirmed by sandwich ELISA. This promising outcome signifies a significant step forward in developing biologic strategies for AOID.