NDLI: Recombinant phospholipase A1 of the outer membrane of psychrotrophic Yersinia pseudotuberculosis: Expression, purification, and characterization

Content Provider	Springer Nature Link
Author	Bakholdina, S. I. Tischenko, N. M. Sidorin, E. V. Isaeva, M. P. Likhatskaya, G. N. Dmitrek, P. S. Kim, N. Yu. Chernikov, O. V. Solov’eva, T. F.
Copyright Year	2016
Abstract	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.
Starting Page	47
Ending Page	57
Page Count	11
File Format	PDF
ISSN	00062979
Journal	Biochemistry (Moscow)
Volume Number	81
Issue Number	1
e-ISSN	16083040
Language	English
Publisher	Pleiades Publishing
Publisher Date	2016-02-02
Publisher Place	Moscow
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Yersinia pseudotuberculosis phospholipase A1 recombinant protein spatial structure modeling Biochemistry Bioorganic Chemistry Microbiology Biomedicine general
Content Type	Text
Resource Type	Article
Subject	Biochemistry Biophysics Geriatrics and Gerontology

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