Production, Purification and Biological Properties of an Escherichia Coli-derived Recombinant Porcine Alpha Interferon

Lefèvre, François; L'Haridon, R.; Borrás‐Cuesta, Francisco; Bonnardière, Claude La

doi:10.1099/0022-1317-71-5-1057

Cited by 39 publications

(18 citation statements)

References 32 publications

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“…The Fluorescein isothiocyanate-labeled annexin V protein was from Bender Med Systems (Boehringer Ingelheim). Human recombinant TNF-␣ (at 100 IU/ml) was from R & D; recombinant porcine IFN-␣ (10 3 IU/l) was kindly provided by C. La Bonnardière (INRA, Jouy-en-Josas, France) (23). Rabbit anti-PARP antibody was from Cell Signalling Technology, and anti-NF-B p65 MAb F6 was from Santa Cruz Biotechnology.…”

Section: Cells and Virusesmentioning

confidence: 99%

African Swine Fever Virus Infection of Porcine Aortic Endothelial Cells Leads to Inhibition of Inflammatory Responses, Activation of the Thrombotic State, and Apoptosis

Vallée

Tait

Powell

2001

J Virol

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Section: Cells and Virusesmentioning

confidence: 99%

African Swine Fever Virus Infection of Porcine Aortic Endothelial Cells Leads to Inhibition of Inflammatory Responses, Activation of the Thrombotic State, and Apoptosis

Vallée

Tait

Powell

2001

J Virol

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“…Pieces of spleen were fixed in 10 % formol in PBS, dehydrated and finally embedded in paraffin (54-56 mC). Rabbit IgGs raised against purified recombinant porcine IFN-α MPA1 (Lefe' vre et al, 1990) were used to stain IFN-α SC in paraffin spleen sections in Tris buffer (50 mM, pH 7n4) with 0n02 % saponin, 0n2 % CaCl # and 1 % heat-inactivated normal porcine serum (NPS). Sections were then incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma), and the IFN-α SC were visualized using the Fast Red substrate (Fast Red TR\Naphthol AS-MX, Sigma).…”

Section: Tissue Of Origin Of Cells Ex Vivo In Vitromentioning

confidence: 99%

In vivo induction of interferon-alpha in pig by non-infectious coronavirus: tissue localization and in situ phenotypic characterization of interferon-alpha-producing cells.

Riffault¹,

Carrat²,

Besnardeau³

et al. 1997

Journal of General Virology

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Section: Introductionmentioning

confidence: 99%

“…Previously, recombinant IFN-α has been successfully expressed in prokaryotes, eukaryotes and baculovirus [2][3][4] . However, the function of the E. coli expressed products was constrained by protein misfolding [3] . The protein expressed in Pichia  İletişim (Correspondence)  +86-551-65123422 (Wang ML), +1-212-305-3310 (Chen J) Fax: 86-551-65123422 (Wang ML), +1-212-305-1262 (Chen J)  microbio@ahmu.edu.cn (Wang ML), jc28@cumc.columbia.edu (Chen J) was readily degradable.…”

Section: Introductionmentioning

confidence: 99%

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

Zhao¹,

Yu²,

Gan³

et al. 2017

Kafkas Univ Vet Fak Derg

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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 Ni 2+ 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 1.1×10 6 IU/ mL in HEp-2/ Vesicular Stomatitis Virus (VSV) titration system and its specific activity reached to 1.0×10 6 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) Rekombinant Domuz İnterferon-α'nın Çözünür Ekspresyonu, Protein Saflaştırması ve Kalite Kontrolü ÖzetBu sunuda rekombinant domuz interferon alfa (rPoIFN-α)'nın Escherichia coli-temelli ekspresyonu ve saflaştırma metodu rapor edilmiştir. PoIFN-α 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-β-D-1-tiogalaktopiyranosid (IPTG) ile uyarmanın ardından rPoIFN-α, bakteri lizatının süpernatantından basit iki basamaklı kromatografi işlemi (Ni 2+ affinite kromatografi ve DEAE anyon değişim kromatografi) kullanılarak saflaştırıldı. rPoIFN-α 48 mg/L kültür oluşumu ve >95% homojenite ile saflaştırıldı. Ürün 6.09 izoelektrik puanına sahip olup bakteriyal endotoksin 1 EU/mg'dan daha azdý. N-ucu amino asit sekansý ve tripsin ile oluþturulan peptit haritasý rPoIFN-α'nın özgünlüğü hakkında ilave kanýt saðladý. rPoIFN-α'nın biyolojik aktivitesi HEp-2/ Vesicular Stomatitis Virus (VSV) titrasyon sisteminde 1.1×10 6 IU/mL olarak tespit edilirken spesifik aktivitesi 1.0×10 6 IU/mg'a ulaştı. Sonuç olarak, pET-32a (+) prokaryotik ekspresyon sistemi kullanılarak biyoaktif rPoIFN-α'nın çözünür formunun yüksek derecede ekspresyonu sağlandı.

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Production, Purification and Biological Properties of an Escherichia Coli-derived Recombinant Porcine Alpha Interferon

Cited by 39 publications

References 32 publications

African Swine Fever Virus Infection of Porcine Aortic Endothelial Cells Leads to Inhibition of Inflammatory Responses, Activation of the Thrombotic State, and Apoptosis

African Swine Fever Virus Infection of Porcine Aortic Endothelial Cells Leads to Inhibition of Inflammatory Responses, Activation of the Thrombotic State, and Apoptosis

In vivo induction of interferon-alpha in pig by non-infectious coronavirus: tissue localization and in situ phenotypic characterization of interferon-alpha-producing cells.

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

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