Isolation and purification of recombinant immunoglobulin light chain variable domains from the periplasmic space of Escherichia coli

Hand, Kieran, Wilkinson, Mark and Madine, Jill ORCID: 0000-0001-9963-5871 (2018) Isolation and purification of recombinant immunoglobulin light chain variable domains from the periplasmic space of Escherichia coli. PLoS One, 13 (10). e0206167-.

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Abstract

Immunoglobulin light chain amyloidosis is the most common form of systemic amyloidosis. However, very little is known about the underlying mechanisms that initiate and modulate the associated protein aggregation and deposition. Model systems have been established to investigate these disease-associated processes. One of these systems comprises two 114 amino acid light-chain variable domains of the kappa 4 IgG family, SMA and LEN. Despite high sequence identity (93%), SMA is amyloidogenic in vivo, but LEN adopts a stable dimer, displaying amyloidogenic properties only under destabilising conditions in vitro. We present here a refined and reproducible periplasmic expression and purification protocol for SMA and LEN that improves on existing methods and provides high yields of pure protein (10-50mg/L), particularly suitable for structural studies that demand highly concentrated and purified proteins. We confirm that recombinant SMA and LEN proteins have structure and dimerization capabilities consistent with the native proteins and employ fluorescence to probe internalization and cellular localization within cardiomyocytes. We propose periplasmic expression and simplified chromatographic steps outlined here as an optimized method for production of these and other variable light chain domains to investigate the underlying mechanisms of light chain amyloidosis. We show that SMA and LEN can be internalised within cardiomyocytes and were observed to localise to the perinuclear area, assessed by confocal microscopy as a possible mechanism for underlying cytotoxicity and pathogenesis associated with amyloidosis.

Item Type:	Article
Uncontrolled Keywords:	Cell Line, Periplasm, Myocytes, Cardiac, Animals, Humans, Rats, Escherichia coli, Immunoglobulin Variable Region, Recombinant Proteins, Microscopy, Confocal, Protein Structure, Secondary, Immunoglobulin Light Chains, Immunoglobulin Light-chain Amyloidosis
Depositing User:	Symplectic Admin
Date Deposited:	24 Oct 2018 07:55
Last Modified:	19 Jan 2023 01:14
DOI:	10.1371/journal.pone.0206167
Open Access URL:	https://doi.org/10.1371/journal.pone.0206167
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3027931