title: A novel expression cassette delivers efficient production of exclusively tetrameric human butyrylcholinesterase with improved pharmacokinetics for protection against organophosphate poisoning.
abstract: Butyrylcholinesterase is a stoichiometric bioscavenger against poisoning by organophosphorus pesticides and nerve agents. The low level of expression and extremely rapid clearance of monomeric recombinant human butyrylcholinesterase (rhBChE) from bloodstream (t½≈2 min) limits its pharmaceutical application. Recently (Ilyushin at al., PNAS, 2013) we described a long-acting polysialylated recombinant butyrylcholinesterase (rhBChE-CAO), stable in the bloodstream, that protects mice against 4.2 LD50 of VR. Here we report a set of modifications of the initial rhBChE expression vector to improve stability of the enzyme in the bloodstream and increase its production in CHO cells by introducing in the expression cassette: (i) the sequence of the natural human PRAD-peptide in frame with rhBChE gene via "self-processing" viral F2A peptide under control of an hEF/HTLV promoter, and (ii) previously predicted in silico MAR 1-68 and MAR X-29 sequences. This provides fully tetrameric rhBChE (4rhBChE) at 70 mg/l, that displays improved pharmacokinetics (t½ = 32 ± 1.2 h, MRT = 43 ± 2 h). 3D Fluorescent visualization and distribution of (125)I-labeled enzyme reveals similar low level 4rhBChE and rhBChE-CAO accumulation in muscle, fat, and brain. Administered 4rhBChE was mainly catabolized in the liver and breakdown products were excreted in kidney. Injection of 1.2 LD50 and 1.1 LD50 of paraoxon to BALB/c and knockout BChE-/- mice pre-treated with 4rhBChE (50 mg/kg) resulted in 100% and 78% survival, respectively, without perturbation of long-term behavior. In contrast, 100% mortality of non-pre-treated mice was observed. The high expression level of 4rhBChE in CHO cells permits consideration of this new expression system for manufacturing BChE as a biopharmaceutical.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho Maklaya 16/10, Moscow, 117997, Russian Federation.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Terekhov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho Maklaya 16/10, Moscow, 117997, Russian Federation.
authors.forename: Ivan
authors.initials: I
authors.lastname: Smirnov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho Maklaya 16/10, Moscow, 117997, Russian Federation; Kazan Federal University, Combinatorial Chemistry and Neurobiology Laboratory, 18 Kremlyovskaya Str, Kazan, 420008, Republic of Tatarstan, Russian Federation.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Bobik
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho Maklaya 16/10, Moscow, 117997, Russian Federation.
authors.forename: Olga
authors.initials: O
authors.lastname: Shamborant
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 8 Lavrentiev Avenue, Novosibirsk, 630090, Russian Federation.
authors.forename: Marina
authors.initials: M
authors.lastname: Zenkova
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 8 Lavrentiev Avenue, Novosibirsk, 630090, Russian Federation.
authors.forename: Elena
authors.initials: E
authors.lastname: Chernolovskaya
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 8 Lavrentiev Avenue, Novosibirsk, 630090, Russian Federation.
authors.forename: Danil
authors.initials: D
authors.lastname: Gladkikh
authors.affiliation: Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 6 Prospekt Nauki, Moscow Region, Pushchino, 142290, Russian Federation.
authors.forename: Arkadii
authors.initials: A
authors.lastname: Murashev
authors.affiliation: Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 6 Prospekt Nauki, Moscow Region, Pushchino, 142290, Russian Federation; Pushchino State Natural-Science Institute, Pushchino, 142290, Russian Federation.
authors.forename: Igor
authors.initials: I
authors.lastname: Dyachenko
authors.affiliation: Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 6 Prospekt Nauki, Moscow Region, Pushchino, 142290, Russian Federation; Pushchino State Natural-Science Institute, Pushchino, 142290, Russian Federation.
authors.forename: Viktor
authors.initials: V
authors.lastname: Palikov
authors.affiliation: Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 6 Prospekt Nauki, Moscow Region, Pushchino, 142290, Russian Federation; Pushchino State Natural-Science Institute, Pushchino, 142290, Russian Federation.
authors.forename: Yulia
authors.initials: Y
authors.lastname: Palikova
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho Maklaya 16/10, Moscow, 117997, Russian Federation.
authors.forename: Vera
authors.initials: V
authors.lastname: Knorre
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho Maklaya 16/10, Moscow, 117997, Russian Federation; Kazan Federal University, Combinatorial Chemistry and Neurobiology Laboratory, 18 Kremlyovskaya Str, Kazan, 420008, Republic of Tatarstan, Russian Federation.
authors.forename: Alexey
authors.initials: A
authors.lastname: Belogurov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho Maklaya 16/10, Moscow, 117997, Russian Federation.
authors.forename: Natalie
authors.initials: N
authors.lastname: Ponomarenko
authors.affiliation: Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S10 2TN, United Kingdom.
authors.forename: G Michael
authors.initials: GM
authors.lastname: Blackburn
authors.affiliation: Kazan Federal University, Neuropharmacology Laboratory, 18 Kremlyovskaya Str, Kazan, 420008, Republic of Tatarstan, Russian Federation; Institute of Structural Biology, DYNAMOP, 71 Avenue des Martyrs CS 10090, 38044, Grenoble Cedex 9, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Masson
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho Maklaya 16/10, Moscow, 117997, Russian Federation. Electronic address: gabibov@ibch.ru.
authors.forename: Alexander
authors.initials: A
authors.lastname: Gabibov
mesh_terms: D000818:Animals; D002091:Butyrylcholinesterase / Q000138:chemical synthesis* / Q000493:pharmacokinetics / Q000494:pharmacology*; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004195:Disease Models, Animal; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D062025:Organophosphate Poisoning / Q000517:prevention & control*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000138:chemical synthesis* / Q000493:pharmacokinetics / Q000494:pharmacology*; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D002091:Butyrylcholinesterase
keywords: Butyrylcholinesterase; Mammalian expression system; Matrix Attachment Region (MAR); Organophosphorus poisoning; Paraoxon