Purification of recombinant human butyrylcholinesterase on Hupresin®

Abstract

Affinity chromatography on procainamide-Sepharose has been an important step in the purification of butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) since its introduction in 1978. The procainamide affinity gel has limitations. In the present report a new affinity gel called Hupresin® was evaluated for its ability to purify truncated, recombinant human butyrylcholinesterase (rHuBChE) expressed in a stably transfected Chinese Hamster Ovary cell line. We present a detailed example of the purification of rHuBChE secreted into 3940 mL of serum-free culture medium. The starting material contained 13,163 units of BChE activity (20.9 mg). rHuBChE was purified to homogeneity in a single step by passage over 82 mL of Hupresin® eluted with 0.1 M tetramethylammonium bromide in 20 mM TrisCl pH 7.5. The fraction with the highest specific activity of 630 units/mg contained 11 mg of BChE. Hupresin® is superior to procainamide-Sepharose for purification of BChE, but is not suitable for purifying native AChE because Hupresin® binds AChE so tightly that AChE is not released with buffers, but is desorbed with denaturing solvents such as 50% acetonitrile or 1% trifluoroacetic acid. Procainamide-Sepharose will continue to be useful for purification of AChE.

Kinetically-controlled mechanism-based isolation of metabolic serine hydrolases in active form from complex proteomes: butyrylcholinesterase as a case study

“…hBChE was most efficiently eluted by a buffer containing tetramethylammonium chloride (4MAC), a weak hBChE activator/inhibitor which has also been used for eluting hBChE from Hupresin. 42 A change of buffer pH did not show signicant effects on the elution efficiency or selectivity of 4MAC (Table 4) and the same proteins were eluted at both pH 7 and pH 5. The amount of eluted enzyme increased upon increasing the concentration of 4MAC up to $1 M and the elution of the protein contaminants followed the same trend (Fig.…”

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

“…Passage of 100 mL human plasma over 2 mL Hupresin yielded 10–15% pure HuBChE [17]. Single-step purification of recombinant HuBChE expressed in insect cells [11] or Chinese Hamster Ovary cells [42] was achieved by chromatography on Hupresin, but not on procainamide gel. Before Hupresin was invented, it was necessary to purify recombinant HuBChE using 3 chromatography steps: procainamide affinity, anion exchange, and a final procainamide affinity chromatography [43].…”

Optical imaging probes for selective detection of butyrylcholinesterase

“…53 In 2012, huprine affinity gel was proposed to be superior due to its higher binding capacity and specificity for BChE compared to procainamide. 24,54,55 Accordingly, the purification of BChE on huprine-Sephraose results in a purity level of 54-90%, while it is only 3-8% for procainamide-Sephrose. 24,56 The mentioned strategies are well defined and employed in numerous studies; however, a cost-effective protocol was developed by Mehrani, in 2003.…”