Affinity Purification of Recombinant Interferon-α on a Mimetic Ligand Adsorbent

Swaminathan, Sathyamangalam; Khanna, Navin

doi:10.1006/prep.1998.1017

Cited by 28 publications

(11 citation statements)

References 16 publications

(20 reference statements)

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“…Therapeutic value of interferons against certain types of tumors such as brain tumors and malignant melanomas caused both increasing interest in these proteins [23] and more focusing on investigations aimed to obtain treated and purified interferons [24]. The purification of human interferons from various sources has been attempted by a variety of methods including metal-chelation, precipitation, cation or anion-exchange, gel filtration, hydrophobic and immunoaffinity chromatography over many years and some protocols have been proposed that yield homogenous protein [25][26][27][28][29].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of human interferon adsorption performance of Cibacron Blue F3GA attached cryogels and interferon purification by using FPLC system

Doğan

Özkara

Sarı

et al. 2012

Journal of Chromatography B

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Section: Introductionmentioning

confidence: 99%

Evaluation of human interferon adsorption performance of Cibacron Blue F3GA attached cryogels and interferon purification by using FPLC system

Doğan

Özkara

Sarı

et al. 2012

Journal of Chromatography B

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“…Since first cloning and expressing IFNα in the 1980s [Baron and Narula, 1990;Goeddel et al, 1980;Mizoguchi et al, 1985;Nagata et al, 1980], several promoter systems have been studied in order to achieve high intracellular expression levels [Babu et al, 2000;Beldarrain et al, 2001;Lim et al, 2000;Neves et al, 2004;Swaminathan and Khanna, 1999]. However, overexpression of recombinant protein in E. coli is prone to the formation of inclusion bodies, which are insoluble and misfolded protein aggregates that are usually biologically inactive.…”

mentioning

confidence: 99%

“…However, overexpression of recombinant protein in E. coli is prone to the formation of inclusion bodies, which are insoluble and misfolded protein aggregates that are usually biologically inactive. Indeed, efforts to efficiently express IFNα in E. coli have been hampered by the formation of intracellular inclusion bodies [Beldarrain et al, 2001;Swaminathan and Khanna, 1999;Villaverde and Carrio, 2003].…”

mentioning

confidence: 99%

Soluble Prokaryotic Expression and Purification of Human Interferon Alpha-2b Using a Maltose-Binding Protein Tag

Jeong²,

Krupa³

et al. 2016

Microb Physiol

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Human interferon alpha-2b (IFNα-2b) has therapeutic applications as an antiviral and antiproliferative drug and has been used for a wide range of indications. Efficient production of IFNα-2b in Escherichia coli has been difficult because the protein tends to form inclusion bodies. This obstacle has garnered interest in efficiently expressing IFNα-2b and overcoming its poor solubility. In this study, seven N-terminal fusion partners - hexahistidine (His6), thioredoxin, glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A, protein disulfide bond isomerase (PDI), and b'a' domain of PDI - were tested for soluble overexpression of codon-optimized IFNα-2b in E. coli. Low temperature increased the expression level of all of the tagged proteins except for the GST fusion. All the tags, except for His6 and GST, improved solubility. We purified IFNα-2b from the MBP-tagged fusion using immobilized metal affinity chromatography and anion exchange chromatography, and obtained a final yield of 7.2 mg from an initial 500-ml culture. The endotoxin level was 0.46 EU/µg. Biological activity was demonstrated using a luciferase assay, which showed a dose-dependent response with a calculated EC₅₀ of 10.3 ± 5.9 pM. Our results demonstrate that using an MBP-tagged fusion is an efficient way to produce pure IFNα-2b.

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“…Among these additives, arginine has been widely used to assist refolding of various recombinant proteins (25)(26)(27)(28). In fact, L-arginine is known as a refolding enhancer and can improve the renaturation yield (25,(29)(30)(31)(32)(33).…”

Section: Introductionmentioning

confidence: 99%

Optimizing Primary Recovery and Refolding of Human Interferon-b from Escherichia coli Inclusion Bodies

Farzaneh¹,

Khodabandeh²,

Arpanaei³

et al. 2014

Iran J Biotech

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Background:The refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives. Objectives: The purpose of this study was optimization of recombinant human interferon-β purification in order to achieve higher efficiency, yield, and a product with a better and more suitable biological activity. Materials and Methods: Triton X-100 and sodium deoxycholate were used to wash the recombinant human interferon-β inclusion bodies prior to solubilization. The inclusion bodies solubilization process was performed by denaturants and reducing agents; guanidine-hydrochloride, urea, β-mercaptoethanol and dithiothritol. Results: The best recovery was obtained in the presence of 0.5% TritonX-100 (v/v). Low concentrations of urea only gave a marginal improvement on the refolding of recombinant human interferon-β. Successful refolding was achieved by gradient elution (decreasing the guanidine-hydrochloride concentration) in the presence of L-arginine. Partial purification was also achieved continuously, and recombinant human interferon-β was recovered with 93.5% purity. The interferon prepared in this project was biologically active and inhibited the replication of vesicular stomatitis virus in Hela cells, when compared to the standard interferon. Conclusions: In this research, the best recovery of inclusion bodies was found at a concentration 0.5 M of Triton X-100, the maximum efficiency of solubility was found in pH 10.5 and the maximum efficiency of refolding was achieved by final buffer containing 2M urea and 0.6 M L-Arg.

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Affinity Purification of Recombinant Interferon-α on a Mimetic Ligand Adsorbent

Cited by 28 publications

References 16 publications

Evaluation of human interferon adsorption performance of Cibacron Blue F3GA attached cryogels and interferon purification by using FPLC system

Evaluation of human interferon adsorption performance of Cibacron Blue F3GA attached cryogels and interferon purification by using FPLC system

Soluble Prokaryotic Expression and Purification of Human Interferon Alpha-2b Using a Maltose-Binding Protein Tag

Optimizing Primary Recovery and Refolding of Human Interferon-b from Escherichia coli Inclusion Bodies

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