title: Towards universal approach for bacterial production of three-finger Ly6/uPAR proteins: Case study of cytotoxin I from cobra N. oxiana.
abstract: Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the 'three-finger' protein superfamily (Ly6/uPAR family) which includes small β-structural proteins (60-90 residues) with high disulfide bond content (4-5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N. oxiana containing additional N-terminal Met-residue (rCTX1) was developed. rCTX1 was produced in the form of E. coli inclusion bodies. Refolding in optimized conditions provided ∼6 mg of correctly folded protein from 1 L of bacterial culture. Cytotoxicity of rCTX1 for C6 rat glioma cells was found to be similar to the activity of wild type CTX1. The milligram quantities of13C,15N-labeled rCTX1 were obtained. NMR study confirmed the similarity of the spatial structures of recombinant and wild-type toxins. Additional Met residue does not perturb the overall structure of the three-finger core. The analysis of available data for different Ly6/uPAR proteins of snake and human origin revealed that efficiency of their folding in vitro is correlated with the number of proline residues in the third loop and the surface area of hydrophobic residues buried within the protein interior. The obtained data indicate that hydrophobic core is important for the folding of proteins with high disulfide bond content. Developed expression method opens new possibilities for structure-function studies of CTX1 and other related three-finger proteins.
authors.affiliation: Biological Faculty, Lomonosov Moscow State University, 119234, Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Street, 117997, Moscow, Russia.
authors.forename: M A
authors.initials: MA
authors.lastname: Shulepko
authors.affiliation: Biological Faculty, Lomonosov Moscow State University, 119234, Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Street, 117997, Moscow, Russia. Electronic address: ekaterina-lyukmanova@yandex.ru.
authors.forename: E N
authors.initials: EN
authors.lastname: Lyukmanova
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Street, 117997, Moscow, Russia; Moscow Institute of Physics and Technology, Institutskiy Pereulok 9, Dolgoprudny, Moscow Region, 141700, Russia.
authors.forename: Z O
authors.initials: ZO
authors.lastname: Shenkarev
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Street, 117997, Moscow, Russia.
authors.forename: P V
authors.initials: PV
authors.lastname: Dubovskii
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Street, 117997, Moscow, Russia.
authors.forename: M V
authors.initials: MV
authors.lastname: Astapova
authors.affiliation: Biological Faculty, Lomonosov Moscow State University, 119234, Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Street, 117997, Moscow, Russia.
authors.forename: A V
authors.initials: AV
authors.lastname: Feofanov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Street, 117997, Moscow, Russia; Moscow Institute of Physics and Technology, Institutskiy Pereulok 9, Dolgoprudny, Moscow Region, 141700, Russia.
authors.forename: A S
authors.initials: AS
authors.lastname: Arseniev
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Street, 117997, Moscow, Russia.
authors.forename: Y N
authors.initials: YN
authors.lastname: Utkin
authors.affiliation: Biological Faculty, Lomonosov Moscow State University, 119234, Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Street, 117997, Moscow, Russia.
authors.forename: M P
authors.initials: MP
authors.lastname: Kirpichnikov
authors.affiliation: Biological Faculty, Lomonosov Moscow State University, 119234, Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Street, 117997, Moscow, Russia.
authors.forename: D A
authors.initials: DA
authors.lastname: Dolgikh
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000494:pharmacology; D045744:Cell Line, Tumor; D004179:Cobra Cardiotoxin Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor; D017815:Elapidae / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D005910:Glioma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D004179:Cobra Cardiotoxin Proteins; D011994:Recombinant Proteins
keywords: Bacterial expression; Nuclear magnetic resonance; Protein refolding; Snake neurotoxin; Three-finger protein