Cloning, Expression, Purification, and Characterization of Soluble Bioactive Recombinant Human Anterior Gradient Homolog 2 - DsRed Monomer Protein in Escherichia coli
Cloning, Expression, Purification, and Characterization of Soluble Bioactive Recombinant Human Anterior Gradient Homolog 2 - DsRed Monomer Protein in Escherichia coli
Bingjie Zhou1, Hitesh Bhagavanbhai Mangukiya1, Siva Bharath Merugu1, Fakhar-un-Nisa Yunus1, Yuchen Fan1, Zhenghua Wu1,* and Dawei Li1,2,*
ABSTRACT
Anterior gradient 2 (AGR2), initially discovered as an estrogen-responsive gene in breast cancer cell lines, is a developmentally regulated gene belonging to the protein disulfide isomerase (PDI) gene family. AGR2 exists in both intracellular and secreted form, it is overexpressed in many types of cancer cells and also detected in extracellular space of solid tumor interstitial fluids. The intracellular AGR2 is critical in protein folding while secreted AGR2 function as a paracrine signal promoting tumor microenvironment formation. Beside cancer progression and drug resistance, the abnormal expression of AGR2 is also involved in diseases like asthma and inflammatory bowel disease. Studies on the role of AGR2 in tumor micro-environment can help potential therapeutic development. Here, we report the generation of a fluorescent functional AGR2 fusion protein as a powerful visual tracking tool to trace the external function of AGR2. This bioactive and auto-fluorescent his-tag recombinant AGR2-DsRed (hAD) protein was constructed by linking human AGR2 C-terminus with the red fluorescent protein derived from Discosoma sp. (DsRed) througha flexible linker. It was expressed successfully in Escherichia coli (E. coli) with optimized strain and cultivation parameters. This protein was expressed and purified by Nickel-nitroacetic acid (Ni-NTA) affinity chromatography. The bioactivity of AGR2 was tested by cell proliferation and wound healing assays, while the auto-fluorescent property of DsRed was confirmed by fluorescence microscopy and immunofluorescence. The potential application of his-AGR2-DsRed was demonstrated by its internalization in fibroblasts. Therefore, the purified recombinant fluorescent protein would be a very useful tool for further investigation of AGR2’s extracellular function and molecular mechanism.
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December 2021
Vol. 53, Iss. 6, Pages 2001-2521
