title: Expression of human AChR extracellular domain mutants with improved characteristics.
abstract: The muscle nicotinic acetylcholine receptor (AChR) has a central role in neuromuscular transmission, and is the major target in the autoimmune disease myasthenia gravis (MG). We created mutants of the extracellular domains (ECDs) of the human α1, β1, δ and ε AChR subunits, whereby their Cys-loop was exchanged for that of the acetylcholine binding protein. The mutants were expressed in Pichia pastoris and had improved solubility resulting in 2- to 43-fold higher expression yields compared to the wild type. An additional mutant was created for the α1 ECD restoring its glycosylation site within the Cys-loop and its α-bungarotoxin binding ability. Furthermore, we constructed dimeric and pentameric concatamers of the mutant ECDs. All concatamers were successfully expressed as soluble secreted proteins, although the pentamers had about 10-fold lower expression than the dimers and were more susceptible to fragmentation. Initial crystallizations with the mutant ECDs were promising, and we reproducibly obtained crystals of the β1 ECD, diffracting at ~12 Å. Further optimization is underway to obtain crystals suitable for high resolution crystallography. The proteins described herein are useful tools in structural studies of the human muscle AChR and can be used in applications requiring high yields such as therapeutic adsorbents for MG autoantibodies.
authors.affiliation: Department of Biochemistry, Hellenic Pasteur Institute, GR11521 Athens, Greece.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Lazaridis
authors.affiliation: Department of Biochemistry, Hellenic Pasteur Institute, GR11521 Athens, Greece.
authors.forename: Paraskevi
authors.initials: P
authors.lastname: Zisimopoulou
authors.affiliation: Department of Biochemistry, Hellenic Pasteur Institute, GR11521 Athens, Greece.
authors.forename: Petros
authors.initials: P
authors.lastname: Giastas
authors.affiliation: Department of Biochemistry, Hellenic Pasteur Institute, GR11521 Athens, Greece.
authors.forename: Kalliopi
authors.initials: K
authors.lastname: Bitzopoulou
authors.affiliation: Department of Biochemistry, Hellenic Pasteur Institute, GR11521 Athens, Greece; Department of Pharmacy, University of Patras, GR26504 Patras, Greece.
authors.forename: Panagiota
authors.initials: P
authors.lastname: Evangelakou
authors.affiliation: Department of Biochemistry, Hellenic Pasteur Institute, GR11521 Athens, Greece.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Sideri
authors.affiliation: Department of Biochemistry, Hellenic Pasteur Institute, GR11521 Athens, Greece; Department of Pharmacy, University of Patras, GR26504 Patras, Greece. Electronic address: tzartos@pasteur.gr.
authors.forename: Socrates J
authors.initials: SJ
authors.lastname: Tzartos
mesh_terms: D000595:Amino Acid Sequence; D002038:Bungarotoxins / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D009132:Muscles / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D009157:Myasthenia Gravis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D002038:Bungarotoxins; D021122:Protein Subunits; D011978:Receptors, Nicotinic
keywords: AChBP; AChR; ECD; MG; MIR; Nicotinic acetylcholine receptor; Pichia pastoris; Protein expression; acetylcholine binding protein; extracellular domain; main immunogenic region; myasthenia gravis; nicotinic acetylcholine receptor; α-Btx; α-bungarotoxin