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
- Elyakov Pacific Institute of Bioorganic Chemistry, Russian Academy of Sciences, Far East Branch, 690022 Vladivostok, Russia
- Far Eastern Federal University, School of Biomedicine, 690950 Vladivostok, Russia
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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The gene fragment of Y. pseudotuberculosis that encodes phospholipase A1 without its signal sequence was amplified using PldFm-Ndel 5’-CAG-CATATGGAAGCAACGATTGAAAAGA-3’ and P1dA-Xhol-Rev 5’-ACTCGAGTTAAAGGACATCGTTCAAC-3’ (restriction sites underlined). PCR amplification was then performed for 5 minutes at 95 °C and then 30 cycles (94 °C, 30 s; 55 °C, 30 s; 72 °C, 40 s), and then 15 minutes at 72 °C. DNA strain 3260 of the Y. pseudotuberculosis was used as the template. From this, the PCR fragment was cloned into the pTZ57R/T vector and then the pIdA gene was subcloned into the pET32b(+) vector at the Ndel (CATATG) and Xhol (CTCGAG) restriction sites before being analyzed using T7 universal primers leading to a plasmid named pET32/mP1dA. E. coli BL21 (DE3) cells were transformed with this plasmid and cultivated at 37 °C for 17 hours under aeration in 2xYTT medium that contained carbenicillin (100 μg/ml). An aliquot of this culture was diluted to 50 fold and then grown to an optical density of 0.4-0.5 at 600 nm in 300 ml of 2xYT medium that contained ampicillin (100 μg/ml). Finally, the recombinant protein expression was induced by IPTG (final conc. 1 mM) for 3 hours before the cells were centrifuged at 6000g at 4 °C and washed with PBS. The wet biomass yield was 2 g/l of culture liquid. Y. pseudotuberculosis rPldA was isolated and purified by isolating the inclusion bodies, dissolving the resulting pellet in 10 ml of buffer A (20 mM Tris-Hcl, ph 8.3; 8 M urea; 0.1 M glycine; 2 mM EDTA), and then sonication and centrifugation at 4500g for 15 minutes.
The inclusion bodies were dissolved in buffer A (20 mM Tris-Hcl, ph 8.3; 8 M urea; 0.1 M glycine; 2 mM EDTA) and passed through a 22 μm filter before chromatography was performed using an HPLC-chromatograph and a column that had been equilibrated with solution (15% acetonitrile, 0.1% TFA by volume). The proteins were extracted with 15-45 (2 ml) and 45-100% (13 ml) acetonitrile gradients at 0.5 ml/min before the fractions were collected. The collected fractions were mixed with a matrix (saturated solution of 3,5-dimethoxy-4-hydroxycinnamic acid (10 mg/ml) in a mixture of acetonitrile with 0.1%^ TFA (1:1, v/v)) and spotted on a MALDI target using the dried-droplet method. The target was analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system.