Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India.
Department of Biotechnology, National Institute of Pharmaceutical Education and Research Guwahati, Changsari, 781101, Guwahati, Assam, India; CSIR-North East Institute of Science & Technology, Jorhat, 785006, Assam, India.
Protein Expr Purif. 2021 Apr;180:105807. doi: 10.1016/j.pep.2020.105807. Epub 2020 Dec 11.
The transcription factor PDX1 is a master regulator essential for proper development of the pancreas, duodenum and antrum. Furthermore, it is an indispensable reprogramming factor for the derivation of human β-cells, and recently, it has been identified as a tumor suppressor protein in gastric cancer. Here, we report the soluble expression and purification of the full-length human PDX1 protein from a heterologous system. To achieve this, the 849 bp coding sequence of the PDX1 gene was first codon-optimized for expression in Escherichia coli (E. coli). This codon-optimized gene sequence was fused to a protein transduction domain, a nuclear localization sequence, and a His-tag, and this insert was cloned into the protein expression vector for expression in E. coli strain BL21(DE3). Next, screening and identification of the suitable gene construct and optimal expression conditions to obtain this recombinant fusion protein in a soluble form was performed. Further, we have purified this recombinant fusion protein to homogeneity under native conditions. Importantly, the secondary structure of the protein was retained after purification. Further, this recombinant PDX1 fusion protein was applied to human cells and showed the ability to enter the cells as well as translocate to the nucleus. This recombinant tool can be used as a safe tool and can potentially replace its genetic and viral forms in the reprogramming process to induce a β-cell-specific transcriptional profile in an integration-free manner. Additionally, it can also be used to elucidate its role in cellular processes and for structural and biochemical studies.
转录因子 PDX1 是胰腺、十二指肠和胃窦正常发育所必需的主要调节因子。此外，它是人类 β 细胞衍生所必需的重编程因子，最近已被确定为胃癌中的肿瘤抑制蛋白。在这里，我们报告了从异源系统中可溶性表达和纯化全长人 PDX1 蛋白。为此，首先对 PDX1 基因的 849 bp 编码序列进行密码子优化，以在大肠杆菌 (E. coli) 中表达。该密码子优化的基因序列与蛋白转导结构域、核定位序列和 His 标签融合，并将该插入物克隆到蛋白表达载体中，在大肠杆菌菌株 BL21(DE3)中表达。接下来，筛选和鉴定合适的基因构建体和最佳表达条件，以获得可溶性形式的这种重组融合蛋白。此外，我们在天然条件下将这种重组融合蛋白纯化至均一性。重要的是，蛋白质的二级结构在纯化后得到保留。此外，这种重组 PDX1 融合蛋白被应用于人类细胞，并显示出进入细胞以及向核易位的能力。这种重组工具可用作安全工具，并且可以潜在地替代其遗传和病毒形式，以非整合方式诱导β细胞特异性转录谱。此外，它还可用于阐明其在细胞过程中的作用以及用于结构和生化研究。
