Purification of human butyrylcholinesterase from frozen Cohn fraction IV-4 by ion exchange and Hupresin affinity chromatography

Abstract

Human butyrylcholinesterase (HuBChE) is being developed as a therapeutic for protection from the toxicity of nerve agents. An enriched source of HuBChE is Cohn fraction IV-4 from pooled human plasma. For the past 40 years, purification of HuBChE has included affinity chromatography on procainamide-Sepharose. The present report supports a new affinity sorbent, Hupresin, for purification of HuBChE from Cohn fraction IV-4. Nine batches of 70–80 kg frozen Cohn fraction were extracted with water, filtered, and chromatographed on 30 L of Q-Ceramic ion exchange sorbent at pH 4.5. The 4% pure Q-eluent was pumped onto 4.2 L Hupresin, where contaminants were washed off with 0.3 M NaCl in 20 mM sodium phosphate pH 8.0, before 99% pure HuBChE was eluted with 0.1 M tetramethylammonium bromide. The average yield was 1.5 g of HuBChE from 80 kg Cohn paste. Recovery of HuBChE was reduced by 90% when the paste was stored at -20°C for 1 year, and reduced 100% when stored at 4°C for 24h. No reduction in HuBChE recovery occurred when paste was stored at -80°C for 3 months or 3 years. Hupresin and procainamide-Sepharose were equally effective at purifying HuBChE from Cohn fraction. HuBChE in Cohn fraction required 1000-fold purification to attain 99% purity, but 15,000-fold purification when the starting material was plasma. HuBChE (P06276) purified from Cohn fraction was a 340 kDa tetramer of 4 identical N-glycated subunits, stable for years in solution or as a lyophilized product.

Chlorpyrifos Oxon Activates Glutamate and Lysine for Protein Cross-linking

“…A reviewer asked whether the nonreducible butyrylcholinesterase dimer 29 could be an organophosphate-induced dimer. We tested this possibility by analyzing gel bands containing the nonreducible butyrylcholinesterase dimer.…”

Rabbit Antidiethoxyphosphotyrosine Antibody, Made by Single B Cell Cloning, Detects Chlorpyrifos Oxon-Modified Proteins in Cultured Cells and Immunopurifies Modified Peptides for Mass Spectrometry

“…The materials used were as follows: Keyhole limpet hemocyanin Megathura crenulata (Calbiochem #374805), trypsin sequencing grade (Promega #V5113), rabbit antidiethoxyphosphotyrosine monoclonal 1C6 (present report), mouse antidiethoxyphosphotyrosine monoclonal 3B9, mouse anti β actin (Thermo Fisher AM4302), antirabbit immunoglobulin G-horseradish peroxidase (IgG-HRP) (Cell Signaling #70745), antimouse IgG-HRP (Cell Signaling #7076S), Halt protease inhibitor cocktail (Thermo Scientific #87786), purified human butyrylcholinesterase prepared in-house, 1-ethyl-3-[3-dimethylaminopropyl]­carbodiimide hydrochloride (EDC) (Fluka #03450), chlorpyrifos oxon (Chem Service MET-11459B), dichlorvos (Chem Service N-11675), trans -retinoic acid (Sigma-Aldrich 554720), CNBr-activated Sepharose 4B (Amersham Biosciences AB #17-981-01), Opti-MEM (Gibco 31985-070), Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Glutamax Gibco 10565-018), radioimmunoprecipitation assay (RIPA) buffer (Pierce #89900), immunoprecipitation (IP) lysis buffer (Pierce 87787 Thermo Fisher Scientific), Clarity Max Western ECL Substrate (Bio-Rad #1705062), Super Signal West Femto maximum sensitivity substrate (Pierce #34095), ECL Super Signal West Dura Substrate Trial Kit (Thermo Scientific 37071), diethoxyphosphotyrosine-modified peptides, YGGFL-OP and RARYEM-OP, for Biacore analysis produced at UNMC using chlorpyrifos oxon to label tyrosine, SH-SY5Y human neuroblastoma cells (ATCC CRL-2266), N2a mouse neuroblastoma cells (ATCC CRL-131), bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific #23225), poly­(vinylidene fluoride) (PVDF) membrane 0.2 μm (Bio-Rad #162-0177), PD-10 desalting column 8.3 mL bed volume (Amersham Pharmacia Biotech #17-0851-01), precast 4–20% gradient polyacrylamide gels (Bio-Rad #4568094), YM-30 regenerated cellulose spin filter (Amicon #42410), and Durapore PVDF-0.45 μm centrifugal filter (Millipore UFC30HV00).…”

“…The materials used were as follows: Keyhole limpet hemocyanin Megathura crenulata (Calbiochem #374805), trypsin sequencing grade (Promega #V5113), rabbit antidiethoxyphosphotyrosine monoclonal 1C6 (present report), mouse antidiethoxyphosphotyrosine monoclonal 3B9, 7 mouse anti β actin (Thermo Fisher AM4302), antirabbit immunoglobulin G-horseradish peroxidase (IgG-HRP) (Cell Signaling #70745), antimouse IgG-HRP (Cell Signaling #7076S), Halt protease inhibitor cocktail (Thermo Scientific #87786), purified human butyrylcholinesterase prepared in-house, 8 ), diethoxyphosphotyrosine-modified peptides, YGGFL-OP and RARYEM-OP, for Biacore analysis produced at UNMC using chlorpyrifos oxon to label tyrosine, 7 SH-SY5Y human neuroblastoma cells (ATCC CRL-2266), N2a mouse neuroblastoma cells (ATCC CRL-131), bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific #23225), poly(vinylidene fluoride) (PVDF) membrane 0.2 μm (Bio-Rad #162-0177), PD-10 desalting column 8.3 mL bed volume (Amersham Pharmacia Biotech #17-0851-01), precast 4−20% gradient polyacrylamide gels (Bio-Rad #4568094), YM-30 regenerated cellulose spin filter (Amicon #42410), and Durapore PVDF-0.45 μm centrifugal filter (Millipore UFC30HV00).…”

Chlorpyrifos Oxon-Induced Isopeptide Bond Formation in Human Butyrylcholinesterase

“…Human butyrylcholinesterase (HuBChE), accession P06276, was purified from frozen Cohn fraction IV-4, in-house [13] and stored as a sterile solution in phosphate buffered saline at 4…”