Kafkas Üniversitesi Veteriner Fakültesi Dergisi 2017 , Vol 23 , Issue 5
Soluble Expression, Protein Purification and Quality Control of Recombinant Porcine Interferon-α
Jun ZHAO1, Hai-yang YU2, Lin GAN2, Yu ZHAO1, Shu-qi LI1, Xiu-le FU1, Ming-li WANG2, Jason CHEN
1Wuhu Overseas Students Pioneer Park, Wuhu, Anhui Province, 241000, CHINA
2Department of Microbiology, Anhui Medical University, Hefei, Anhui Province, 230032, CHINA
3Anhui JiuChuan Biotech Co., Ltd, Wuhu, Anhui Province, 241007, CHINA
4Department of Pathology & Cell Biology, Columbia University, New York, 10032, USA
DOI : 10.9775/kvfd.2017.17372 Herein, we reported an Escherichia coli-based expression and purification method of recombinant porcine interferon alpha (rPoIFN-α). PoIFN-α 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-β-D-1-thiogalactopyranoside (IPTG), rPoIFN-α 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-α 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-α. The biological activity of rPoIFN-α was 1.1×106 IU/ mL in HEp-2/ Vesicular Stomatitis Virus (VSV) titration system and its specific activity reached to 1.0×106 IU/mg. In conclusion, we obtained high-level expression of a soluble form of bioactive rPoIFN-α by using pET-32a (+) prokaryotic expression system. Keywords : Soluble expression, Protein purification, Quality control, Porcine interferon-α, Vesicular Stomatitis Virus (VSV)