title: Purification of phosphinothricin acetyltransferase using Reactive brown 10 affinity in a single chromatography step.
abstract: The expression of phosphinothricin N-acetyltransferase (PAT) protein in transgenic plants confers tolerance to the herbicide glufosinate. To enable the characterization of PAT protein expressed in plants, it is necessary to obtain high purity PAT protein from the transgenic grain. Because transgenically expressed proteins are typical present at very low levels (i.e. 0.1-50 μg protein/g grain), a highly specific and efficient purification protocol is required to purify them. Based on the physicochemical properties of PAT, we developed a novel purification method that is simple, time-saving, inexpensive and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein was purified to homogeneity from cottonseed with high recovery efficiency. As expected, the Reactive brown 10-produced PAT was enzymatically active. Other applications of the method on protein expression and purification, and development of PAT enzymatic inhibitors were also discussed.
authors.affiliation: Monsanto Company, 800 North Lindbergh Blvd, St. Louis, MO 63167, United States. cunxi.wang@monsanto.com
authors.forename: Cunxi
authors.initials: C
authors.lastname: Wang
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Lee
authors.forename: Kathleen S
authors.initials: KS
authors.lastname: Crowley
authors.forename: Erin
authors.initials: E
authors.lastname: Bell
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry / Q000302:isolation & purification*; D002845:Chromatography / Q000379:methods*; D004396:Coloring Agents; D007475:Ion Exchange Resins; D014227:Triazines
chemical_list: D004396:Coloring Agents; D007475:Ion Exchange Resins; D014227:Triazines; C053802:Procion Brown MX 5BR; D000123:Acetyltransferases; C056243:phosphinothricin N-acetyltransferase
keywords: Affinity chromatography; Cottonseed; Phosphinothricin N-acetyltransferase; Reactive brown 10; Reactive dye