Recombinant phospholipase A^sub 1^ of the outer membrane of psychrotrophic Yersinia pseudotuberculosis: Expression, purification, and characterization

The pldA gene encoding membrane-bound phospholipase A^sub 1^ of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase A^sub 1^ (rPldA) was isolated from inclusion bodies dissolved in 8 M urea by two-stage chromatography (ion-exchange and gel-filtra...

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Veröffentlicht in:Biochemistry (Moscow) 2016-01, Vol.81 (1), p.47
Hauptverfasser: Bakholdina, S I, Tischenko, N M, Sidorin, E V, Isaeva, M P, Likhatskaya, G N, Dmitrenok, P S, Kim, N Yu, Chernikov, O V, Solov'eva, T F
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Sprache:eng
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Zusammenfassung:The pldA gene encoding membrane-bound phospholipase A^sub 1^ of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase A^sub 1^ (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 A^sub 1^ 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 A^sub 1^ were constructed, and functionally important amino acid residues of the enzyme were determined. Structural differences between phospholipases A^sub 1^ from Y. pseudotuberculosis and E. coli, which can affect the functional activity of the enzyme, were revealed.
ISSN:0006-2979
1608-3040
DOI:10.1134/S0006297916010053