Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration.

作者: Kristina Witte , Anette Kaiser , Peter Schmidt , Victoria Splith , Lars Thomas

DOI: 10.1515/HSZ-2013-0120

关键词:

摘要: In vitro folding of G protein-coupled receptors into a detergent environment represents promising strategy for obtaining sufficient amounts functional receptor molecules structural studies. Typically, these preparations exhibit poor long-term stability especially at the required high protein concentration. Here, we report protocol stabilization Escherichia coli-expressed and subsequently folded neuropeptide Y type 2. We identified free cysteines in as one major reason intermolecular aggregation. Therefore, six out eight cysteine residues were mutated to alanine or serine without any significant loss functionality demonstrated cell culture models. Furthermore, disulfide bond between remaining two was irreversibly formed by applying oxidative folding. Applying this strategy, functionally Y2 could be increased 20 days concentration 15 μm micelle consisting 1,2-diheptanoyl-sn-glycero-3-phosphocholine n-dodecyl-s-D-maltoside.

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