Purification and refolding of Escherichia coli-expressed recombinant human interleukin-2

The expression of rhIL‐2 (recombinant human interleukin‐2) in bacteria results in the formation of insoluble inclusion‐body aggregates. These aggregates were first solubilized under denaturing conditions (sodium phosphate buffer solution containing 8 M urea and 10 mM 2‐mercaptoethanol) and then puri...

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Bibliographic Details
Published in:Biotechnology and applied biochemistry 2010-04, Vol.55 (4), p.209-214
Main Authors: Esfandiar, Samaneh, Hashemi-Najafabadi, Sameereh, Shojaosadati, Seyed Abbas, Sarrafzadeh, Shokuh Aazam, Pourpak, Zahra
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biotechnology
Chromatography, Affinity - methods
Escherichia
Escherichia coli - genetics
Escherichia coli - metabolism
Fundamental and applied biological sciences. Psychology
His6-tagged recombinant human interleukin-2 (rhIL-2)
Humans
immobilized metal-ion-affinity chromatography (IMAC)
Inclusion Bodies - metabolism
Interleukin-2 - biosynthesis
Interleukin-2 - chemistry
Interleukin-2 - genetics
Interleukin-2 - isolation & purification
lymphocyte transformation test (LTT)
metal-affinity chromatography purification
Ni2+-nitrilotriacetate-agarose (Ni-NTA-agarose)
Nitrilotriacetic Acid - analogs & derivatives
Nitrilotriacetic Acid - chemistry
Organometallic Compounds - chemistry
Protein Folding
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
refolding
Solubility
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Summary:The expression of rhIL‐2 (recombinant human interleukin‐2) in bacteria results in the formation of insoluble inclusion‐body aggregates. These aggregates were first solubilized under denaturing conditions (sodium phosphate buffer solution containing 8 M urea and 10 mM 2‐mercaptoethanol) and then purified using IMAC (immobilized metal‐ion‐affinity chromatography). IMAC was used to capture rhIL‐2. The protein was gradually refolded on the column by a gradient elution (8 M to 0 M urea) in the presence of 10% (v/v) glycerol. Glycerol was used to prevent protein aggregation during the refolding step. Using this method, rhIL‐2 was collected at 97% purity and its activity was measured by the lymphocyte transformation test. The measured activity was identical with commercial human interleukin‐2.
ISSN:0885-4513
1470-8744
DOI:10.1042/BA20090256