The [(bathophenanthroline) 3:Fe 2+] complex as an aromatic non-polymeric medium for purification of human lactoferrin.

Thisara Jayawickrama Withanage, Mitra Lal, Hagit Salem, Olga Krichevski, Ellen Wachtel, Guy Patchornik

نتاج البحث: نشر في مجلةمقالةمراجعة النظراء

ملخص

We describe a non-chromatographic, ligand-free platform for the efficient purification of recombinant human lactoferrin (LF). The platform consists of a [metal:chelator] 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) 3:Fe 2+] was determined to be the most efficient. LF is recovered with high yield (∼90%, 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 [cation:π] interactions between the lysine and arginine residues of LF with the fully aromatic [(batho) 3:Fe 2+] complexes. The use of [metal:chelator] complex aggregates is demonstrated to provide an economical and efficient avenue for LF purification.

اللغة الأصليةالإنجليزيّة
رقم المقال465218
الصفحات (من إلى)465218
دوريةJournal of Chromatography
مستوى الصوت1732
المعرِّفات الرقمية للأشياء
حالة النشرنَشْر مسبق في الإنترنت - 29 يوليو 2024

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