ni 2 affinity column  (TaKaRa)

Journal: Poultry Science

Article Title: Recombinant LAB vector engineered to secrete chicken IFN-λ3 induces an early but sustained activation of antiviral host responses against AIV infection

doi: 10.1016/j.psj.2025.105973

Figure Lengend Snippet: Panel A: Cloning, expression, purification, and secondary structure determination of recombinant ChIFN-λ3 protein in E. coli (a) Colony PCR of the transformed E. coli Rosetta 2 (DE3) cells harbouring pHis-ChIFN-λ3 plasmid showing the amplified product (∼495 bp) corresponding to the size of the ChIFN-λ3 gene (black arrow). (b) SDS PAGE analysis of IPTG-induced rChIFN-λ3 protein expression in the cell lysate of E. coli cells (white arrow) with a molecular weight of ∼21 kDa. (c) Different elution fractions of Ni 2+ -NTA affinity column-purified Histidine-tagged rChIFN-λ3 protein. (d, e) Western blot analysis of purified rChIFN-λ3 protein probed with mouse monoclonal anti-His antibody, as well as mouse polyclonal antibody, respectively, confirms the detection of the desired protein (∼ 21 kDa). (f) Experimental CD spectra of purified rChIFN-λ3 protein in Tris buffer. The CD signal (Δε) was plotted against wavelength (nm) in the far-UV range (180-260 nm), suggesting that the ChIFN-λ3 protein has a higher percentage (%) of alpha helices. Panel B: Assessment of cell cytotoxicity of rChIFN-λ3 protein in E. coli The cell cytotoxicity of rChIFN-λ3 protein was determined in (a) CEF cells and (b) CEICs, suggesting the non-toxic nature of the protein (CC 50 >50 μg/mL; CC 50 >150 μg/mL) to the primary chicken cells. Panel C: Confocal and flow cytometry analysis to determine the rChIFN-λ3 protein binding to primary CEF cells (a) Confocal microscopy images confirm the binding ability of rChIFN-λ3 protein to primary CEF cells. The mice polyclonal anti-ChIFN-λ3 antibody was used as primary antibody (1:100 dilution), while FITC labelled goat anti-rabbit IgG (H + L) was used as secondary antibody (1:500). All images were captured in a Leica SP8 confocal microscope using an oil immersion 60 × objective (NA 1.4) with 1.2 × zoom, and the LAS-X software was then used to acquire and process the images (scale bar =10 μM). The experiment was performed twice under similar conditions. (b) Flow cytometric analysis reveals a clear shift in the CEF cell population when charged with ChIFN-λ3 protein, further suggesting a dose-dependent binding of the protein to the cells. The data represent the mean ± SE of four independent experiments ( n = 4); asterisks indicate statistically significant differences (* P ≤ 0.05) between the antibody control and rChIFN-λ3 treatment groups.

Article Snippet: The supernatant was collected, and the protein was purified by the Ni 2+ -NTA affinity column (Ni 2+ -NTA Superflow, Qiagen, USA).

Techniques: Cloning, Expressing, Purification, Recombinant, Transformation Assay, Plasmid Preparation, Amplification, SDS Page, Molecular Weight, Affinity Column, Western Blot, Circular Dichroism, Flow Cytometry, Protein Binding, Confocal Microscopy, Binding Assay, Microscopy, Software, Control