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KMID : 1094720110160040688
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Biotechnology and Bioprocess Engineering
2011 Volume.16 No. 4 p.688 ~ p.688
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Expression, purification, and characterisation of nesiritide using an E. coli expression system
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Rao Laxmi S.
Niphadkar Milind P.
Paliwal Dinesh
Shekhawat Rakesh
Khare Aruna G.
Uma S.
Thakur Priti
Chutke Anjali
Surlikar Neelesh
Samant Radhika
Zawar Sagar
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Abstract
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Nesiritide, the recombinant human brain natriuretic peptide, is involved in the regulation of cardiovascular homeostasis and has been approved for treatment of patients with congestive heart failure. We prepared a synthetic cDNA construct of Nesiritide to generate a fusion protein with an affinity handle and 41 amino acid peptide of β-galactosidase. The fusion protein was expressed mainly in the inclusion bodies and accounted for approximately 20% of total cellular protein. After purification by Ni-IDA affinity chromatography and renaturation, the fusion protein was cleaved with purified recombinant enterokinase. Nesiritide was purified by pH precipitation/ion exchange chromatography followed by source phenyl chromatography to obtain protein with > 99% purity (determined by RPHPLC) and a mass of 3,464 Daltons. The potency (ED50) of the purified protein was equivalent to that of Natrecor (Innovator formulation). Analytical methods were developed to identify oxidised, reduced and other related impurities. The expression strategy described in this work allows the convenient generation of high yield Nesiritide and enabled ease of purification.
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KEYWORD
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brain natriuretic peptide, nesiritide, expression, bioprocess, purification, characterization
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