Journal
JOURNAL OF BIOTECHNOLOGY
Volume 168, Issue 4, Pages 666-675Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2013.09.011
Keywords
Recombinant human lactoferrin; Gene expression; Antioxidant; Inflammation
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Funding
- National Institute of Allergy and Infectious Diseases [R42AI051050-05, PO1 AI062885]
- National Institute of General Medicine [5R42GM079810-04]
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Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins. The aim of this study was to scale-up expression and purification of rhLF in a CHO expression system, verify its glycan primary structure, and assess its biological properties in cell culture models. A stable CHO cell line producing >200 mg/L of rhLF was developed and established. rhLF was purified by a single-step cation-exchange chromatography procedure. The highly homogenous rhLF has a molecular weight of approximately 80 kDa. MALDI-TOF mass spectrometric analysis revealed N-linked, partially sialylated glycans at two glycosylation sites, typical for human milk LF. This novel rhLF showed a protective effect against oxidative stress in a similar manner to its natural counterpart. In addition, rhLF revealed a modulatory effect on cellular redox via upregulation of key antioxidant enzymes. These data imply that the CHO-derived rhLF is fully compatible with the native molecule, thus it has promise for human therapeutic applications. (C) 2013 Elsevier B.V. All rights reserved.
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