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-filt...

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Bibliographic Details
Published in:Biochemistry (Moscow) 2016-01, p.47
Main Authors: Bakholdina, S.I, Tischenko, N.M, Sidorin, E.V, Isaeva, M.P, Likhatskaya, G.N, Dmitrenok, P.S, Kim, N. Yu, Chernikov, O.V, Soloveva, T.F
Format: Article
Language:English
Subjects:
Gene expression
Genetic aspects
Health aspects
Observations
Phospholipases
Recombinant proteins
Yersinia
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Summary: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. DOI: 10.1134/S0006297916010053 Key words: Yersinia pseudotuberculosis, phospholipase [A.sub.1], recombinant protein, spatial structure modeling
ISSN:0006-2979
DOI:10.1134/S0006297916010053