The [(bathophenanthroline)3:Fe2+] complex as an aromatic non-polymeric medium for purification of human lactoferrin.
J Chromatogr A
; 1732: 465218, 2024 Jul 29.
Article
em En
| MEDLINE
| ID: mdl-39106663
ABSTRACT
We describe a non-chromatographic, ligand-free platform for the efficient purification of recombinant human lactoferrin (LF). The platform consists of a [metalchelator] complex precipitate in the presence of osmotically active polyethylene glycol 6000 (PEG-6000). Purification is achieved in three stages. Following formation of the complex, LF is captured under neutral conditions by the aggregated complexes (Step I), a washing step follows (Step II) and then, (Step III) LF is extracted in pure form with 100 mM tribasic Na citrate buffer (pH 7). Of the four complexes investigated, [bathophenanthroline (batho)3Fe2+] was determined to be the most efficient. LF is recovered with high yield (â¼90%, by densitometry) and purity (≥97%, by SDS polyacrylamide gel electrophoresis (SDS-PAGE)) from an artificial contamination background comprising E. coli lysate proteins. Purified LF is demonstrated to be monomeric by dynamic light scattering (DLS); to preserve its native secondary structure by circular dichroism (CD) spectroscopy; and, as apo-LF, to efficiently inhibit bacterial growth. Process yield is not affected by a 45-fold increase in LF concentration from 0.2 to 9 mg/mL. We provide evidence that protein capture relies on [cationπ] interactions between the lysine and arginine residues of LF with the fully aromatic [(batho)3Fe2+] complexes. The use of [metalchelator] complex aggregates is demonstrated to provide an economical and efficient avenue for LF purification.
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01-internacional
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MEDLINE
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En
Ano de publicação:
2024
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Article