title: Recombinant Phospholipase A1 of the Outer Membrane of Psychrotrophic Yersinia pseudotuberculosis: Expression, Purification, and Characterization.
abstract: The pldA gene encoding membrane-bound phospholipase A1 of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase A1 (rPldA) was isolated from inclusion bodies dissolved in 8 M urea by two-stage chromatography (ion-exchange and gel-filtration chromatography) as an inactive monomer. The molecular mass of the rPldA determined by MALDI-TOF MS was 31.7 ± 0.4 kDa. The highly purified rPldA was refolded by 10-fold dilution with buffer containing 10 mM Triton X-100 and subsequent incubation at room temperature for 16 h. The refolded rPldA hydrolyzed 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in the presence of calcium ions. The enzyme exhibited maximal activity at 37°C and nearly 40% of maximal activity at 15°C. The phospholipase A1 was active over a wide range of pH from 4 to 11, exhibiting maximal activity at pH 10. Spatial structure models of the monomer and the dimer of Y. pseudotuberculosis phospholipase A1 were constructed, and functionally important amino acid residues of the enzyme were determined. Structural differences between phospholipases A1 from Y. pseudotuberculosis and E. coli, which can affect the functional activity of the enzyme, were revealed.
authors.affiliation: Elyakov Pacific Institute of Bioorganic Chemistry, Russian Academy of Sciences, Far East Branch, Vladivostok, 690022, Russia. sibakh@mail.ru.
authors.forename: S I
authors.initials: SI
authors.lastname: Bakholdina
authors.forename: N M
authors.initials: NM
authors.lastname: Tischenko
authors.forename: E V
authors.initials: EV
authors.lastname: Sidorin
authors.forename: M P
authors.initials: MP
authors.lastname: Isaeva
authors.forename: G N
authors.initials: GN
authors.lastname: Likhatskaya
authors.forename: P S
authors.initials: PS
authors.lastname: Dmitrenok
authors.forename: N Yu
authors.initials: NY
authors.lastname: Kim
authors.forename: O V
authors.initials: OV
authors.lastname: Chernikov
authors.forename: T F
authors.initials: TF
authors.lastname: Solov'eva
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D054466:Phospholipases A1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D015011:Yersinia pseudotuberculosis / Q000201:enzymology*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D011994:Recombinant Proteins; D054466:Phospholipases A1