On-column Chromatographic Refolding Of Recombinant Human Interferon-γ Inclusion Bodies

As a cytokine, interferon-gamma (IFN-γ) is a homodimeric protein that plays an essential role in cell-mediated immune responses to viral and mycobacterial infections. Recombinant human IFN-γ (rhIFN-γ) is a good approach to meet the requirement of clinical therapy in large amounts. However, rhIFN-γ is expressed as IBs in E.coli and the renaturation of the protein is a bottleneck of the process.Here, we reported some on-column chromatographic processes for the refolding and purification of rhIFN-y produced in E.coli. SDS-PAGE was performed for protein analysis and rhIFN-γ activity was determined by cytopathic effect (CPE) method as well as ABS-ELISA method.The rhIFN-γ produced in the E.coli host after induction at 42℃ and accumulated as inclusion bodies (IBs). Yield of the rhIFN-γ IBs was approximately 1.6g/L culture. After washed with the detergent buffer including 50 mmol/L Tris-HCl and 0.5% Triton X-100, the purity of IBs was up to 85%.Ion exchange chromatography (IEC) with urea gradient was performed as protein refolding system. The process of refolding and purification could be integrated together by IEC. The specific activity of rhIFN-γ was 7.5 ×105 IU/mg and the protein yield was 54% under such experiment conditions: protein loading 2.5mg, flow rate 0.5mL/min, urea gradient length 3.4CV and final urea concentration 2mol/L. The purity of rhIFN-γ determined by SES-PAGE was above 95%.Refolding processes by hydrophobic interaction chromatography (HIC) with no urea gradient and linear decreased urea gradient were proposed. Under the optimum conditions in the refolding with linear decreased urea gradient, the activity yield of rhIFN-γ was 6.5 times of that in the dilution refolding process and the protein mass yield was up to 36%.Minichaperone (sht GroEL191-345) was produced in 3.7L agitated bioreactor. The rhIFN-y IBs were refolded effectively by a combined column with immobilized minichaperone at Ni-NTA agarose, which was connected with a short SEC column. As a result, the activity yield of rhIFN-γ was 4.2 times of that in the dilution refolding and the protein yield was 77%.