Optimization of the primary recovery of human interferon alpha2b from Escherichia coli inclusion bodies.
The human interferon alpha2b (hu-IFNalpha2b) gene was cloned in Escherichia coli JM109(DE3) and the recombinant protein was expressed as cytoplasmic inclusion bodies (IB). The present work discusses the recovery of hu-IFNalpha2b IB from the E. coli cells. An optimized protocol is proposed based on the sequential evaluation of recovery steps and parameters: (i) cell disruption, (ii) IB recovery and separation from cell debris, (iii) IB washing, and (iv) IB solubilization. Parameters such as hu-IFNalpha2b purity and recovery yield were measured after each step. The optimized recovery protocol yielded 60% of hu-IFNalpha2b with a purity of up to 80%. The protein was renatured at high concentration after recovery and it was found to display biological activity.
Valente
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CA
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. claudivalente@yahoo.com
Monteiro
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Cabral
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JM
Fevereiro
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Prazeres
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D000818:Animals; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D014508:Urea / Q000737:chemistry; D014709:Vero Cells
D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D014508:Urea