Production and characterization of active recombinant interleukin‐12/eGFP fusion protein in stably‐transfected DF1 chicken cells

Abstract

The adjuvant activity of chicken interleukin-12 (chIL-12) protein has been described as similar to that of mammalian IL-12. Recombinant chIL-12 can be produced using several methods, but chIL-12 production in eukaryotic cells is lower than that in prokaryotic cells. Stimulating compounds, such as dimethyl sulfoxide (DMSO), can be added to animal cell cultures to overcome this drawback. In this study, we constructed a cell line, DF1/chIL-12 which stably expressed a fusion protein, chIL-12 and enhanced green fluorescent protein (eGFP) connected by a (G4 S)3 linker sequence. Fusion protein production was increased when cells were cultured in the presence of DMSO. When 1 × 10(6) DF1/chIL-12 cells were inoculated in a T-175 flask containing 30 mL of media, incubated for 15 h, and further cultivated in the presence of 4% DMSO for 48 h, the production of total fusion protein was mostly enhanced compared with the production of total fusion protein by using cell lysates induced with DMSO at other concentrations. The concentrations of the unpurified and purified total fusion proteins in cell lysates were 2,781 ± 2.72 ng mL(-1) and 2,207 ± 3.28 ng mL(-1) , respectively. The recovery rate was 79%. The fusion protein stimulated chicken splenocytes to produce IFN-γ, which was measured using an enzyme-linked immunosorbent assay, in the culture supernatant, indicating that treating DF1/chIL-12 cells with DMSO or producing chIL-12 in a fusion protein form does not have adverse effects on the bioactivity of chIL-12.

Characterization of functionally active interleukin‐18/eGFP fusion protein expression during cell cycle phases in recombinant chicken DF1 Cells

“…To identify the fusion protein production, both the culture supernatant and cell lysate were examined by western‐blotting. The results indicated that fusion protein in the culture supernatant was not detected (data not shown), similar to those of our previous studies, in which rchIL‐12 or IL‐18 synthesized by rFPV/chIL‐12 or −18 10 or chIL‐12/eGFP fusion protein was evident as a cell‐associated protein. Conversely, a major protein band with an estimated size of 55 kDa, which is consistent with the expected size of the fusion protein, was detected in the cell lysate of DF1/chIL‐18a, because this protein was recognized by antibodies against the FLAG peptide (Figure C, lane 2), eGFP (Figure D, lane 2), and the echIL‐18 (Figure E, lane 2) .…”

“…Such protein production was lower, in comparison to the aforementioned results. This could be because the fusion protein, chIL‐18/eGFP, rather than a single chIL‐18 was synthesized in this study, rendering possible the less effective expression of such a large protein, as previously described …”

“…Stably transfected DF1 chicken cells expressing active recombinant chIL‐18/eGFP fusion were generated as described previously . The chIL‐18‐encoding region was cloned into an eGFP gene that was inserted into a pcDNA3.1 vector to generate a pchIL‐18 insertion vector.…”

“…The insertion vector was designated pchIL‐18 (Supporting Information Figure S1C). Subsequently, the same procedures to those for generating chIL‐12/eGFP fusion protein were used for generating the stably transfected DF1 cells expressing chIL‐18/eGFP fusion protein.…”

“…To determine the concentration of the total fusion protein, serial tenfold dilutions of known concentrations of the eGFP‐FLAG protein were prepared to generate a standard curve for further calculating the fusion protein concentration. Cell lysates were prepared from DF1/chaIL‐18a cells cultured with or without aphidicolin for 6 days and 100 μL of the cell lysates were used for determining amounts of the total fusion protein by conducting a sandwich enzyme‐linked immunosorbent assay (s‐ELISA) as described …”