Jun ZHAO, Hai-yang YU, Lin GAN, Yu ZHAO, Shu-qi LI, Xiu-le FU, Ming-li WANG, Jason CHEN

Soluble Expression, Protein Purification and Quality Control of Recombinant Porcine Interferon-α

Bu sunuda rekombinant domuz interferon alfa (rPoIFN-?)'nın Escherichia coli-temelli ekspresyonu ve saflaştırma metodu rapor edilmiştir. PoIFN-? kodlayan sekansı pMD18-T vektör içine klonlandı ve sonrasında standart rekombinant DNA teknikleri kullanılarak pET-32a (+) vektör içine subklonlandı ve elde edilen plazmid BL21(DE3) kompetan hücreler içine nakledildi. İzopropil-?-D-1-tiogalaktopiyranosid (IPTG) ile uyarmanın ardından rPoIFN-?, bakteri lizatının süpernatantından basit iki basamaklı kromatografi işlemi (Ni2+ affinite kromatografi ve DEAE anyon değişim kromatografi) kullanılarak saflaştırıldı. rPoIFN-? 48 mg/L kültür oluşumu ve >95% homojenite ile saflaştırıldı. Ürün 6.09 izoelektrik puanına sahip olup bakteriyal endotoksin 1 EU/mg'dan daha azd?. N-ucu amino asit sekans? ve tripsin ile olu?turulan peptit haritas? rPoIFN-?'nın özgünlüğü hakkında ilave kan?t sa?lad?. rPoIFN-?'nın biyolojik aktivitesi HEp-2/ Vesicular Stomatitis Virus (VSV) titrasyon sisteminde 1.1×106 IU/mL olarak tespit edilirken spesifik aktivitesi 1.0×106 IU/mg'a ulaştı. Sonuç olarak, pET-32a (+) prokaryotik ekspresyon sistemi kullanılarak biyoaktif rPoIFN-?'nın çözünür formunun yüksek derecede ekspresyonu sağlandı

Rekombinant Domuz İnterferon-α’nın Çözünür Ekspresyonu, Protein Saflaştırması ve Kalite Kontrolü

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

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