Narayan Gloria, Agrawal Akriti, Sen Plaboni, Nagotu Shirisha, Thummer Rajkumar P
Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India.
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India.
Protein J. 2023 Dec;42(6):766-777. doi: 10.1007/s10930-023-10143-3. Epub 2023 Aug 8.
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）是一种关键的转录因子，在胚胎发育过程中参与胰腺生成，在成体中，它对β细胞的增殖和存活至关重要。此外，PAX4在人类黑色素瘤中还作为一种肿瘤抑制蛋白发挥作用。本研究展示了生物活性重组人PAX4转录因子的产生。首先，将插入片段（两端带有标签的PAX4蛋白编码序列）克隆到表达载体中，构建出pET28a(+)-HTN-PAX4和pET28a(+)-PAX4-NTH基因构建体，然后将它们转化到大肠杆菌中进行表达。从1.2升大肠杆菌培养物中分别纯化出产量约为3.15毫克和0.83毫克的HTN-PAX4和PAX4-NTH融合蛋白。此外，还证明了PAX4融合蛋白的二级结构保留情况及其内化哺乳动物细胞及其细胞核的潜力。使用各种检测方法（细胞迁移、细胞增殖和细胞周期检测）研究了这些融合蛋白的生物活性，证明其作为肿瘤抑制蛋白发挥作用。因此，这种大分子有望有助于理解人类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。
