Abstract
Palmitoylation refers to the attachment of palmitate to protein cysteine residues and assists with protein localization to the plasma membrane. Like protein phosphorylation, palmitoylation is often a dynamic process, and it involves the attachment of the palmitate by palmitoyl transferases and removal by palmitoyl protein thioesterase (PPTs) or acyl protein thioesterases (APTs). Currently, the techniques to study palmitoylation are difficult to perform and involve the use of harsh chemical reagents like hydroxylamine (HAM) to depalmitate proteins, often leading to destruction of the proteins being studied. Recombinant, purified PPT/APTs could be used as an alternative reagent to depalmitate proteins and also to investigate their individual specificities in depalmitoylation reactions. To purify APTs and PPTs, the coding sequences of human variants were PCR amplified, cloned and expressed to produce either GST or MBP‐tagged fusion proteins. Expressed proteins have been analyzed using SDS‐PAGE and bands of expected molecular mass have been identified. Enzyme activity is currently being tested with the substrate 4‐methylumbelliferyl‐6‐thiopalmitoyl‐b‐glucoside (MU‐6S‐palm‐bGlc) in a fluorogenic enzyme assay, with the goal of eventually using the fusion proteins in depalmitolyation reactions involving palmitoylated protein substrates.Support or Funding InformationThis work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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