Soluble Expression, Protein Purification and Quality Control of Recombinant Porcine Interferon-α
| Autor: | Xiu-le Fu, Lin Gan, Jun Zhao, Ming-Li Wang, Zhao Yu, Shu-qi Li, Jason Chen, Hai-yang Yu |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
General Veterinary Chemistry Veterinary medicine Expression protein law.invention 03 medical and health sciences 030104 developmental biology Biochemistry law protein purification Interferon α vesicular stomatitis virus (vsv) SF600-1100 Recombinant DNA soluble expression quality control porcine interferon-α |
| Zdroj: | Kafkas Universitesi Veteriner Fakultesi Dergisi, Vol 23, Iss 5, Pp 825-829 (2017) |
| ISSN: | 1300-6045 |
| DOI: | 10.9775/kvfd.2017.17372 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|