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Cloning of Novel Leukocytic X5 ARHGEF18 Proteoform for Functional Characterization

Rho GTPases are bio‐molecular switches in cells with activation depending on guanine nucleotide exchange factors (ARHGEFs) that facilitate GDP (guanosine diphosphate) release so GTP (guanosine triphosphate) can bind to the GTPase. When GTP is bound, GTPases are activated and contribute to cell signaling which includes regulating diverse responses such as gene transcription, cytoskeletal rearrangements, cell growth and motility. Eosinophils undergo a rapid and profound morphological change after cytokine stimulation. ARHGEF18 is abundant in eosinophils, and based on its activity in other cell types could be responsible for the formation of actin stress fibers allowing for the cell's dramatic polarization. However, our data indicates that eosinophil ARHGEF18 has an N‐terminal extension of 349 residues (part of LOC100996504 (LOC1009), which is annotated in UniProt as a unique protein) to comprise a 1364‐residue protein that has never been characterized. This project encompasses the cloning, expression, purification, and characterization of LOC1009‐ARHGEF18 (LOCGEF) and other constructs as a precursor to assessing the functional significance of LOCGEF for cytoskeleton rearrangement. The sequence of interest was created using PCR amplification of the complementary DNA sequences created from the eosinophil cellular mRNA with reverse transcription. Four separate constructs were created, one containing the entire sequence (LOCGEF), canonical ARHGEF18 without the LOC1009 sequence (p114; 1015 residues), and two sequences, one composed of LOC1009 exons 1–10 (LOC ex10; 322 residues), the other LOC1009 exons 1–11 (LOC ex11; 346 residues). The PCR product was restricted and ligated into a bacterial plasmid with a histidine tag encoded at the N‐terminus. E. coli were transformed with these plasmids using the heat shock method and plated onto kanamycin containing media. Those with the ability to grow on the media contained plasmids with the construct sequences. Bacterial expression of LOC ex10 and LOC ex11 was induced with Isopropyl β‐D‐1‐thiogalactopyranoside and the resulting proteins purified using a Ni‐NTA column. Purified LOC ex10 protein was tested in ELISA and detected using rabbit antibodies raised against the LOC1009 portion of the construct (anti‐LOC100996504 IgG; Abcam 185114). LOC ex11 has yet to be tested in ELISA. Plasmids for p114 and LOCGEF also have been created but protein yield for p114 and LOCGEF was poor in the E. coli expression system. The next step is to express these proteins in a eukaryotic system. The characterization of LOCGEF is important in understanding eosinophil cytoskeletal rearrangement needed for immune response. The function of the N‐terminal extension of LOCGEF and how it correlates with functions of the shorter isoform of ARHGEF18 previously studied in other cell types are unknown. The creation of these proteins and constructs will enable structural characterization, antibody binding, and discovery of binding partners in pull‐down assays.