Soluble Expression, Protein Purification And Quality Control Of Recombinant Porcine Interferon-Alpha

Herein, we reported an Escherichia coli-based expression and purification method of recombinant porcine interferon alpha (rPoIFN-alpha). PoIFN-alpha coding sequence was cloned into pMD18-T vector and then subcloned into pET-32a (+) vector using standard recombinant DNA techniques and the resulting plasmid was transformed into BL21(DE3) competent cells. After induction with isopropyl-alpha-D-1-thiogalactopyranoside (IPTG), rPoIFN-a was purified from the supernatant of the bacteria lysate using a simple two-step chromatography process consisting of a Ni2+ affinity chromatography and a DEAE anion exchange chromatography. rPoIFN-a was purified to > 95% homogeneity with a yield of 48 mg/L of culture. It has isoelectic point of 6.09 and bacterial endotoxin was less than 1 EU/mg. N-terminal amino acid sequence and the peptide map digested by trypsin provided additional evidence for the authenticity of rPoIFN-alpha. The biological activity of rPoIFN-alpha was 1.1x10(6) IU/mL in HEp-2/Vesicular Stomatitis Virus (VSV) titration system and its specific activity reached to 1.0x10(6) IU/mg. In conclusion, we obtained high-level expression of a soluble form of bioactive rPoIFN-a by using pET-32a (+) prokaryotic expression system.