Recombinant Expression and Purification of F-box and Leucine-rich Repeat Protein 5 (FBXL5) using a Prokaryotic Expression System

Fatima Joy C Cruz; Tomohide Saio; Takeshi Uchida; Koichiro Ishimori

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Recombinant Expression and Purification of F-box and Leucine-rich Repeat Protein 5 (FBXL5) using a Prokaryotic Expression System

Author: Fatima Joy C. Cruz ¹ ; Tomohide Saio ² ; Takeshi Uchida ² ; Koichiro Ishimori ²
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1. Department of Physical Sciences and Mathematics, College of Arts and Sciences, University of the Philippines Manila, Manila, Philippines
2. Structural Chemistry Laboratory, Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Japan
Publication Type:Journal Article
Keywords: Iron metabolism; Ubiquitin ligase
MeSH: Leucine-Rich Repeat Proteins
From: Philippine Journal of Health Research and Development 2022;26(CAS Issue):41-57
CountryPhilippines
Language:English
Abstract: Background:The F-box and Leucine-rich Repeat Protein 5 (FBXL5), a member of the E3 ligases, is considered to be the central iron sensor in mammals. The cryo-EM structure of FBXL5 in complex with IRP2 and SKP1 was reported by Wang et.al. in 2020. Surprisingly, a 2Fe-2S cluster seemed to be responsible for the iron-sensing capability of FBXL5.
Objectives:To further explore the mechanism of its regulation, it is important to study the interaction of FBXL5 with other proteins under regulated conditions so we attempted to express FBXL5 in the hopes of studying its interaction with IRPs in vitro.
Methodology:Plasmids were constructed to express FBXL5 in Escherichia coli expression hosts. Purification of an MBP-fused FBXL5 and GST-fused FBXL5 were performed using affinity chromatography. Peptide Mass Fingerprinting, Circular Dichroism spectroscopy, and SEC-MALS were employed to analyze the purified MBP-FBXL5. GST-FBXL5 was also used in a pull-down assay with Iron Regulatory Protein 1 (IRP1).
Results:We are successful in expressing and partially purifying full-length FBXL5 using E. coli with the aid of a protein tag, the maltose binding protein (MBP) tag. However, cleavage of the protein tag resulted in decreased stability of FBXL5 as shown in SEC-MALS data. CD spectroscopy showed consistent secondary structure of FBXL5. A preliminary pull-down assay of GST-FBXL5 with IRP1 showed that IRP1 displayed interaction with the recombinant GST-FBXL5.
Conclusion:FBXL5, a 78-kDa mammalian protein was overexpressed in a prokaryotic expression system made stable by a fusion protein. The interaction of GST-FBXL5 with IRP1 also shows that it is possible to study their interaction in vitro.
Full text:569-1404-2-PB1.pdf