title: His-tag truncated butyrylcholinesterase as a useful construct for in vitro characterization of wild-type and variant butyrylcholinesterases.
abstract: Human butyrylcholinesterase (BChE) can scavenge and thereby provide protection against various toxic esters, including organophosphate-based chemical warfare agents and the recreational drug cocaine. It is currently being used in molecular evolution studies to generate novel enzymes with improved ability to hydrolyze toxic ester compounds. Currently, the most commonly used purification strategies for recombinant BChE enzymes involve using affinity resins based on small molecule interactions with the enzyme's substrate binding site. However, as BChE variants are discovered and developed, a generic purification protocol that is insensitive to amino acid substitutions is necessary. In the current manuscript, an expression vector encoding a C-terminal truncation and a His₆-tag was designed for BChE and used to express recombinant "wild-type" enzyme and two variants (i.e., G117H BChE and G117H/E197Q BChE). All the three His₆-tagged enzymes were successfully purified via metal-affinity columns using similar procedures with good recovery. Steady-state kinetic parameters were determined for each enzyme, and values were compared to those obtained with the corresponding non-truncated non-His₆-tagged enzymes. Rates of inhibition by echothiophate, a model compound for organophosphate-based pesticides, and rates of oxime-mediated reactivation after inhibition with a nerve agent model compound were also determined for selected enzymes. Rates of spontaneous reactivation from ETP inhibition were determined for the G117H variants. In all instances examined, truncation of the C-terminus of BChE and introduction of a His₆-tag had no significant effects on the observed kinetic parameters, making this a highly useful construct for in vitro characterization of wild-type and variant BChEs.
authors.affiliation: Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121, USA. eralph@hbri.org
authors.forename: Erik C
authors.initials: EC
authors.lastname: Ralph
authors.forename: Longkuan
authors.initials: L
authors.lastname: Xiang
authors.forename: John R
authors.initials: JR
authors.lastname: Cashman
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
mesh_terms: D019943:Amino Acid Substitution; D002091:Butyrylcholinesterase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D002619:Chemical Warfare Agents / Q000378:metabolism; D015870:Gene Expression; D006639:Histidine / Q000235:genetics; D006801:Humans; D009842:Oligopeptides / Q000235:genetics; D009943:Organophosphorus Compounds / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D002619:Chemical Warfare Agents; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D006639:Histidine; D002091:Butyrylcholinesterase