Protocol for the purification of the ABC transporter OpuA and reconstitution into Nanodiscs

Abstract

This protocol has been optimized in the context of:
Van Den Noort, M., Drougkas, P., Paulino, C., and Poolman, B. The substrate-binding domains of the osmoregulatory ABC importer OpuA physically interact. eLife 12:RP90996
https://doi.org/10.7554/eLife.90996.1

Lactococcus lactis OpuA is a type I ABC importer. It employs two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver it to the transmembrane domains for transport. OpuA detects osmotic stress through alterations in internal ionic strength and is further modulated by the second messenger cyclic-di-AMP. It exists as a tetrameric protein complex composed of two distinct subunits: two cytosolic OpuAA proteins featuring a nucleotide-binding domain (NBD) and a cyclic-di-AMP-sensing (CBS) domain, and two OpuABC proteins comprising a transmembrane domain (TMD), a scaffold domain, and an extracellular single substrate-binding domain (SBD). The oligomeric assembly exhibits modest stability, with a notable portion of OpuA complex dissociating during purification. Consequently, modifications were made to the purification protocol across various parameters to minimize OpuA complex dissociation.

Protocol References

Van Den Noort, M., Drougkas, P., Paulino, C., and Poolman, B. The substrate-binding domains of the osmoregulatory ABC importer OpuA physically interact. eLife 12:RP90996
https://doi.org/10.7554/eLife.90996.1