Adapting recombinant bacterial alkaline phosphatase for nucleotide exchange of small GTPases

Author:

Frank,Peter

Hong, Min

Higgins, Brianna

Perkins, Shelley

Taylor, Troy

Wall,Vanessa

Drew,Matt

Waybright, Timothy

Gillette,Bill

Esposito,Dom

Messing, Simon
Author Address

NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA., NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA. Electronic address: simon.messing@nih.gov.,

Abstract:

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20-30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment. Copyright © 2024. Published by Elsevier Inc.

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