Expression of recombinant swine ferritin heavy chain with enhanced solubility in Escherichia coli and simplified purification of ferritin nanoparticles

IF 1.4 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS Protein expression and purification Pub Date : 2025-03-12 DOI:10.1016/j.pep.2025.106700
M.S.B.W.T.M. Nipuna Sudaraka Tennakoon , Kyoung-Ho Lee , Hyun-Jin Shin
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Abstract

Ferritin is a versatile biomolecule used in various medical applications such as drug delivery, vaccines, biological imaging, and diagnostics. The purity and concentration of the ferritin nanoparticles are crucial for achieving excellent outcomes. In this study, we expressed and purified the recombinant swine ferritin heavy chain (rsFTH) as a new candidate for recombinant ferritin nanoparticles. We generated two types of plasmids that can express rsFTH in mammalian and prokaryotic systems. The myc-tagged rsFTH expressed in the mammalian system was purified and ferritin nanoparticles were validated using dynamic light scattering (DLS) and transmission electron microscopy (TEM). A prokaryotic expression system was used to produce rsFTH on a large scale. Protein expression was optimized in Escherichia coli BL21 under varying temperatures and IPTG conditions, and solubility was enhanced by incubation at 25 °C for 18–22 h in auto-induction media, resulting in approximately >50 % protein content in the soluble fraction compared with the pellet. Protein purification was achieved using His-tag affinity chromatography and dialysis with Tris-HCl buffer, yielding adequately pure rsFTH without any apparent protein aggregates. SDS-PAGE and Western blot analysis confirmed the expected molecular weight of rsFTH, and Native-PAGE demonstrated polymerization into higher molecular weight forms. Particle size analysis of purified rsFTH revealed a mean diameter of 15.5 nm, with transmission electron microscopy (TEM) imaging confirming spherical ferritin particles with an iron core. These results suggest that rsFTH can be efficiently expressed and purified in both mammalian and bacterial systems, and has potential applications in nanotechnology and biotechnology.
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来源期刊
Protein expression and purification
Protein expression and purification 生物-生化研究方法
CiteScore
3.70
自引率
6.20%
发文量
120
审稿时长
32 days
期刊介绍: Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.
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Editorial Board Screening and Functional Characterization of Nanobodies Targeting the Transferrin Receptor. Expression of recombinant swine ferritin heavy chain with enhanced solubility in Escherichia coli and simplified purification of ferritin nanoparticles Rational designation and characterization of a novel humanized collagen capable of self-assembling into triple helix and fibrils with D-period High-purity preparation and biophysical characterization of OX40-TC in lipid nanodiscs
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