High-efficient bacterial production of human ApoA-I amyloidogenic variants

Research output: Contribution to journalArticle

Abstract

Apolipoprotein A-I (ApoA-I)-related amyloidosis is a rare disease caused by missense mutations in the APOA1 gene. These mutations lead to protein aggregation and abnormal accumulation of ApoA-I amyloid fibrils in heart, liver, kidneys, skin, nerves, ovaries, or testes. Consequently, the carriers are at risk of single- or multi-organ failure and of need of organ transplantation. Understanding the basic molecular structure and function of ApoA-I amyloidogenic variants, as well as their biological effects, is, therefore, of great interest. However, the intrinsic low stability of this type of proteins makes their overexpression and purification difficult. To overcome this barrier, we here describe an optimized production and purification procedure for human ApoA-I amyloidogenic proteins that efficiently provides between 46 mg and 91 mg (depending on the protein variant) of pure protein per liter of Escherichia coli culture. Structural integrity of the amyloidogenic and native ApoA-I proteins were verified by circular dichroism spectroscopy and intrinsic fluorescence analysis, and preserved functionality was demonstrated by use of a lipid clearance assay as well as by reconstitution of high-density lipoprotein (HDL) particles. In conclusion, the use of the described high-yield protein production system to obtain amyloidogenic ApoA-I proteins, and their native counterpart, will enable molecular and cellular experimental studies aimed to explain the molecular basis for this rare disease.

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Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Cell and Molecular Biology

Keywords

  • amyloidosis, apolipoprotein A-I, HDL, high-density lipoprotein, high-yield protein production
Original languageEnglish
Pages (from-to)2101-2109
Number of pages9
JournalProtein Science
Volume27
Issue number12
Publication statusPublished - 2018
Publication categoryResearch
Peer-reviewedYes