Paired box 4 (PAX4) is a pivotal transcription factor involved in pancreatogenesis during embryogenesis, and in adults, it is key for β-cell proliferation and survival. Additionally, PAX4 also functions as a tumor suppressor protein in human melanomas. The present study demonstrates the production of bioactive recombinant human PAX4 transcription factor. At first, the inserts (PAX4 protein-coding sequence having tags at either ends) were cloned in an expression vector to give rise to pET28a(+)-HTN-PAX4 and pET28a(+)-PAX4-NTH genetic constructs, and these were then transformed into Escherichia coli (E. coli) for their expression. The HTN-PAX4 and PAX4-NTH fusion proteins produced were purified with a yield of ~ 3.15 mg and ~ 0.83 mg, respectively, from 1.2 L E. coli culture. Further, the secondary structure retention of the PAX4 fusion proteins and their potential to internalize the mammalian cell and its nucleus was demonstrated. The bioactivity of these fusion proteins was investigated using various assays (cell migration, cell proliferation and cell cycle assays), demonstrating it to function as a tumor suppressor protein. Thus, this macromolecule can prospectively help understand the function of human PAX4 in cellular processes, disease-specific investigations and direct cellular reprogramming.

配对盒 4 (PAX4) 是参与胚胎发生过程中胰腺发生的关键转录因子，在成人中，它是 β 细胞增殖和存活的关键。此外，PAX4 还在人类黑色素瘤中充当肿瘤抑制蛋白。本研究展示了具有生物活性的重组人 PAX4 转录因子的生产。首先，将插入片段（两端都有标签的 PAX4 蛋白编码序列）克隆到表达载体中，以产生 pET28a(+)-HTN-PAX4 和 pET28a(+)-PAX4-NTH 基因构建体，这些是然后转化到大肠杆菌（E. coli）中进行表达。从 1.2 L大肠杆菌培养物中纯化产生的 HTN-PAX4 和 PAX4-NTH 融合蛋白，产量分别为约 3.15 mg 和约 0.83 mg 。此外，还证明了 PAX4 融合蛋白的二级结构保留及其内化哺乳动物细胞及其细胞核的潜力。使用各种测定（细胞迁移、细胞增殖和细胞周期测定）研究了这些融合蛋白的生物活性，证明其具有肿瘤抑制蛋白的功能。因此，这种大分子可以前瞻性地帮助了解人类 PAX4 在细胞过程、疾病特异性研究和直接细胞重编程中的功能。