Cloning, expression, purification and characterization of patatin, a novel phospholipase A

Authors


  • Definition: phospholipids are named according to the IUBMB recommmendations as follows: PCho, phosphocholine; diC6PCho, 1,2-dihexanoyl-sn-glycero-3-phosphocholine; diC7PCho, 1,2-diheptanoyl-sn-glycero-3-phosphocholine; diC8PCho, 1,2-dioctanoyl-sn-glycero-3-phosphocholine; diC9PCho, 1,2-dinonanoyl-sn-glycero-3-phosphocholine; diC10PCho, 1,2-didecanoyl-sn-glycero-3-phosphocholine; diC12PCho, 1,2-didodecanoyl-sn-glycero-3-phosphocholine; diC16PCho, 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine; C18lysoPCho, 1-octadecanoyl-2-lyso-sn-glycero-3-phosphocholine; C8thioglycolPCho, 2-octanoylthio-ethane-1-phosphocholine; C12thioglycolPCho, 2-dodecanoylthio-ethane-1-phosphocholine; C16thioglycolPCho, 2-hexadecanoylthio-ethane-1-phosphocholine; diC8dithioPCho, rac-1,2-dioctanoyldithiopropyl-3-phosphocholine

M. R. Egmond, Department of Enzymology and Protein Engineering, Padualaan 8, De Uithof, 3584 CH Utrecht, the Netherlands

Abstract

Patatin is the major protein constituent of potato tubers and displays broad esterase activity. The native enzyme actually belongs to a highly homologous multigene family of vacuolar glycoproteins. From these, the patB2 patatin gene was selected and cloned into pUC19 without its signal sequence but with an N-terminal histidine-tag. This patatin was overexpressed under the control of the lac promotor in Escherichia coli strain DH5α. The protein was recovered as inclusion bodies, folded into its native state by solubilization in urea and purified to homogeneity. Starting with one gram of inclusion bodies, 19 mg of pure and active recombinant patatin was isolated, with even higher specific activity than the glycosylated wild-type patatin purified from potato tubers. The purified enzyme showed esterolytic activity with p-nitrophenylesters dissolved in Triton X-100 micelles. The activity of patatin on p-nitrophenylesters with different carbon chain lengths showed an optimum for p-nitrophenylesters with 10 carbon atoms. Besides general esterolytic activity, the pure enzyme was found to display high phospholipase A activity in particular with the substrates 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC8PCho) (127 U·mg−1) and 1,2-dinonanoyl-sn-glycero-3-phosphocholine (diC9PCho) (109 U·mg−1).

Recently, the structure of human cytosolic PLA2 (cPLA2) was solved, showing a novel Ser-Asp active site dyad [1]. Based on a partial sequence alignment of patatin with human cPLA2, we propose that patatin contains a similar active site dyad. To verify this assumption, conserved Ser, Asp and His residues in the family of patatins have been modified in patatin B2. Identification of active site residues was based on the observation of correctly folded but inactive variants. This led to the assignment of Ser54 and Asp192 as the active site serine and aspartate residues in patatin B2, respectively.

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