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Recombinant Phospholipase A1 of the Outer Membrane of Psychrotrophic Yersinia pseudotuberculosis: Expression, Purification, and Characterization

S. I. Bakholdina1*, N. M. Tischenko1, E. V. Sidorin1, M. P. Isaeva1,2, G. N. Likhatskaya1, P. S. Dmitrenok1, N. Yu. Kim1, O. V. Chernikov1, and T. F. Solov’eva1

1Elyakov Pacific Institute of Bioorganic Chemistry, Russian Academy of Sciences, Far East Branch, 690022 Vladivostok, Russia; fax: + 7 (423) 231-4050; E-mail: sibakh@mail.ru

2Far Eastern Federal University, School of Biomedicine, 690950 Vladivostok, Russia; fax: + 7 (423) 245-7687

* To whom correspondence should be addressed.

Received February 24, 2015; Revision received July 14, 2015
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.
KEY WORDS: Yersinia pseudotuberculosis, phospholipase A1, recombinant protein, spatial structure modeling

DOI: 10.1134/S0006297916010053