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

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S. I. Bakholdina^1*, N. M. Tischenko¹, E. V. Sidorin¹, M. P. Isaeva^1,2, 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

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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.

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KEY WORDS: Yersinia pseudotuberculosis, phospholipase A₁, recombinant protein, spatial structure modeling

DOI: 10.1134/S0006297916010053

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