Efficient production of human interleukin‐3 from Escherichia coli using protein disulfide isomerase b'a' domain

• Published in: Biotechnology journal 2024-05, Vol.19 (5), p.e2300581-n/a
• Main Authors: Nguyen, Thi Kieu Oanh, Ryu, Dayoung, Nguyen, Minh Quan, Ta, Huynh Kim Khanh, Vu, Thi Luong, Choe, Han
• Format: Article
• Language: English
• Subjects: Cell Line, Tumor; E. coli expression; Escherichia coli - genetics; Escherichia coli - metabolism; Humans; Interleukin-3 - genetics; Interleukin-3 - metabolism; interleukin‐3 (IL3); Maltose-Binding Proteins - genetics; Maltose-Binding Proteins - metabolism; PDIb'a' domain; Protein Disulfide-Isomerases - genetics; Protein Disulfide-Isomerases - metabolism; protein solubility; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Solubility; TF1 cell proliferation
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.202300581

Human interleukin‐3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N‐terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various E. coli strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H‐PDIb'a'‐IL3 was purified using two chromatographic methods, and the subsequent removal of the H‐PDIb'a' tag yielded high‐purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF‐1 cells revealed a unique two‐step stimulation pattern for both purified IL3 and the H‐PDIb'a'‐IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies. Graphical and Lay Summary The IL3 construct was genetically fused with fusion tags and a TEV protease cleavage site. Subsequently, these fusion proteins were introduced into suitable E. coli strains, leading to their expression in a soluble form within the cytoplasm. Initial purification was carried out using immobilized metal affinity chromatography (IMAC) with a HisTrap column, followed by obtaining the target proteins through TEV protease cleavage. Further purification was achieved through Ion Exchange Chromatography (IEC) utilizing a HiTrap Q column, based on the isoelectric point (pI) value. The structural characterization of the purified IL3 was determined through LC‐MS/MS assay. Finally, a two‐step stimulation of the purified IL3 was conducted on human leukemia cells (TF‐1 cells).