title: Receptor-binding domain of ephrin-A1: production in bacterial expression system and activity.
abstract: Eph receptor tyrosine kinases and their ligands, the ephrins, perform an important regulatory function in tissue organization, as well as participate in malignant transformation of cells. Ephrin-A1, a ligand of A class Eph receptors, is a modulator of tumor growth and progression, and the mechanism of its action needs detailed investigation. Here we report on the development of a system for bacterial expression of an ephrin-A1 receptor-binding domain (eA1), a procedure for its purification, and its renaturation with final yield of 50 mg/liter of culture. Functional activity of eA1 was confirmed by immunoblotting, laser scanning confocal microscopy, and flow cytometry. It is shown that monomeric non-glycosylated receptor-binding domain of ephrin-A1 is able to activate cellular EphA2 receptors, stimulating their phosphorylation. Ligand eA1 can be used to study the features of ephrin-A1 interactions with different A class Eph receptors. The created expression cassette is suitable for the development of ligands with increased activity and selectivity and experimental systems for the delivery of cytotoxins into tumor cells that overexpress EphA2 or other class A Eph receptors.
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia. office@ibch.ru
authors.forename: O V
authors.initials: OV
authors.lastname: Nekrasova
authors.forename: G V
authors.initials: GV
authors.lastname: Sharonov
authors.forename: R V
authors.initials: RV
authors.lastname: Tikhonov
authors.forename: P M
authors.initials: PM
authors.lastname: Kolosov
authors.forename: M V
authors.initials: MV
authors.lastname: Astapova
authors.forename: S A
authors.initials: SA
authors.lastname: Yakimov
authors.forename: A I
authors.initials: AI
authors.lastname: Tagvey
authors.forename: A A
authors.initials: AA
authors.lastname: Korchagina
authors.forename: O V
authors.initials: OV
authors.lastname: Bocharova
authors.forename: A N
authors.initials: AN
authors.lastname: Wulfson
authors.forename: A V
authors.initials: AV
authors.lastname: Feofanov
authors.forename: M P
authors.initials: MP
authors.lastname: Kirpichnikov
mesh_terms: D003001:Cloning, Molecular; D036382:Ephrin-A1 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D061986:MCF-7 Cells; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D036104:Receptor, EphA2 / Q000378:metabolism; D036081:Receptors, Eph Family / Q000378:metabolism*; D012995:Solubility; D014867:Water / Q000737:chemistry
chemical_list: D036382:Ephrin-A1; D014867:Water; D036104:Receptor, EphA2; D036081:Receptors, Eph Family