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Expression, Purification and Initial Characterization of Functional alpha(1)-Microglobulin (A1M) in Nicotiana benthamiana

Carlsson, Magnus and Kristiansson, Amanda and Bergwik, Jesper and Kanagarajan, Selvaraju and Bulow, Leif and Akerstrom, Bo and Zhu, Li-Hua (2020). Expression, Purification and Initial Characterization of Functional alpha(1)-Microglobulin (A1M) in Nicotiana benthamiana. Frontiers in Plant Science. 11 , 593773
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Abstract

alpha(1)-Microglobulin (A1M) is a small glycoprotein that belongs to the lipocalin protein family. A major biological role of A1M is to protect cells and tissues against oxidative damage by clearing free heme and reactive oxygen species. Because of this, the protein has attracted great interest as a potential pharmaceutical candidate for treatment of acute kidney injury and preeclampsia. The aim of this study was to explore the possibility of expressing human A1M in plants through transient gene expression, as an alternative or complement to other expression systems. E. coli, insect and mammalian cell culture have previously been used for recombinant A1M (rA1M) or A1M production, but these systems have various drawbacks, including additional complication and expense in refolding for E. coli, while insect produced rA1M is heavily modified with chromophores and mammalian cell culture has been used only in analytical scale. For that purpose, we have used a viral vector (pJL-TRBO) delivered by Agrobacterium for expression of three modified A1M gene variants in the leaves of N. benthamiana. The results showed that these modified rA1M protein variants, A1M-NB1, A1M-NB2 and A1M-NB3, targeted to the cytosol, ER and extracellular space, respectively, were successfully expressed in the leaves, which was confirmed by SDS-PAGE and Western blot analysis. The cytosol accumulated A1M-NB1 was selected for further analysis, as it appeared to have a higher yield than the other variants, and was purified with a yield of ca. 50 mg/kg leaf. The purified protein had the expected structural and functional properties, displaying heme-binding capacity and capacity of protecting red blood cells against stress-induced cell death. The protein also carried bound chromophores, a characteristic feature of A1M and an indicator of a capacity to bind small molecules. The study showed that expression of the functional protein in N. benthamiana may be an attractive alternative for production of rA1M for pharmaceutical purposes and a basis for future research on A1M structure and function.

Authors/Creators:Carlsson, Magnus and Kristiansson, Amanda and Bergwik, Jesper and Kanagarajan, Selvaraju and Bulow, Leif and Akerstrom, Bo and Zhu, Li-Hua
Title:Expression, Purification and Initial Characterization of Functional alpha(1)-Microglobulin (A1M) in Nicotiana benthamiana
Series Name/Journal:Frontiers in Plant Science
Year of publishing :2020
Volume:11
Article number:593773
Number of Pages:14
Publisher:FRONTIERS MEDIA SA
ISSN:1664-462X
Language:English
Publication Type:Journal article
Article category:Scientific peer reviewed
Version:Published version
Copyright:Creative Commons: Attribution 4.0
Full Text Status:Public
Subjects:(A) Swedish standard research categories 2011 > 4 Agricultural Sciences > 404 Agricultural Biotechnology > Plant Biotechnology
Keywords:α(1)-microglobulin, N, benthamiana, agroinfiltration, stress protection, pharmaceutical use, heme binding
URN:NBN:urn:nbn:se:slu:epsilon-p-110094
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-p-110094
Additional ID:
Type of IDID
DOI10.3389/fpls.2020.593773
Web of Science (WoS)000600624000001
ID Code:21446
Faculty:LTV - Fakulteten för landskapsarkitektur, trädgårds- och växtproduktionsvetenskap
Department:(LTJ, LTV) > Department of Plant Breeding (from 130101)
Deposited By: SLUpub Connector
Deposited On:21 Jan 2021 14:03
Metadata Last Modified:21 Jan 2021 14:11

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