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Jens Lagerstedt

Associate professor

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

Author

  • Rita Del Giudice
  • Jens O. Lagerstedt

Summary, in English

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.

Department/s

  • Medical Protein Science
  • EXODIAB: Excellence in Diabetes Research in Sweden

Publishing year

2018

Language

English

Pages

2101-2109

Publication/Series

Protein Science

Volume

27

Issue

12

Document type

Journal article

Publisher

The Protein Society

Topic

  • Cell and Molecular Biology

Keywords

  • amyloidosis
  • apolipoprotein A-I
  • HDL
  • high-density lipoprotein
  • high-yield protein production

Status

Published

Research group

  • Medical Protein Science

ISBN/ISSN/Other

  • ISSN: 0961-8368