Purification of recombinant human butyrylcholinesterase on Hupresin®

© 2018 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

Cell-free H-cluster Synthesis and [FeFe] Hydrogenase Activation: All Five CO and CN− Ligands Derive from Tyrosine

[FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO) and two cyanide (CN−) ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG) are presumably responsible for assembling and installing the H-cluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN− ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation

High-Yield Expression of Heterologous [FeFe] Hydrogenases in Escherichia coli

BACKGROUND: The realization of hydrogenase-based technologies for renewable H(2) production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases. PRINCIPAL FINDINGS: In this report, we describe an improved Escherichia coli-based expression system capable of producing 8-30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H(2) evolution with rates comparable to those of enzymes isolated from their respective native organisms. SIGNIFICANCE: The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H(2)-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments

Purification of recombinant human butyrylcholinesterase on Hupresin®

© 2018 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

Hupresin, a new affinity resin to purify butyrylcholinesterase

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Crystallization and preliminary X-ray diffraction data for the aconitase form of human iron-regulatory protein 1

Two crystal forms of the aconitase version of recombinant human IRP1 are reported

8-Hydroxyquinolylnitrones as multifunctional ligands for the therapy of neurodegenerative diseases

We describe the development of quinolylnitrones (QNs) as multifunctional ligands inhibiting cholinesterases (ChEs: acetylcholinesterase and butyrylcholinesterase-hBChE) and monoamine oxidases (hMAO-A/B) for the therapy of neurodegenerative diseases. We identified QN 19, a simple, low molec-ular weight nitrone, that is readily synthesized from commercially available 8-hydroxyquinoline-2-carbaldehyde. Quinolylnitrone 19 has no typical pharmacophoric element to suggest ChE or MAO inhi-bition, yet unexpectedly showed potent inhibition of hBChE (IC50 = 1.06 +/- 0.31 nmol/L) and hMAO-B (IC50 = 4.46 +/- 0.18 mmol/L). The crystal structures of 19 with hBChE and hMAO-B provided the struc-tural basis for potent binding, which was further studied by enzyme kinetics. Compound 19 acted as a free radical scavenger and biometal chelator, crossed the blood-brain barrier, was not cytotoxic, and showed neuroprotective properties in a 6-hydroxydopamine cell model of Parkinson's disease. In addition, in vivo studies showed the anti-amnesic effect of 19 in the scopolamine-induced mouse model of AD without adverse effects on motoric function and coordination. Importantly, chronic treatment of double transgenic APPswe-PS1dE9 mice with 19 reduced amyloid plaque load in the hippocampus and cortex of female mice, underscoring the disease-modifying effect of QN 19. 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)