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Titolo:
Cloning, expression, purification and characterization of patatin, a novelphospholipase A
Autore:
Hirschberg, HJHB; Simons, JWFA; Dekker, N; Egmond, MR;
Indirizzi:
Univ Utrecht, Dept Enzymol & Prot Engn, CBLE, NL-3584 CH Utrecht, Netherlands Univ Utrecht Utrecht Netherlands NL-3584 CH 3584 CH Utrecht, Netherlands
Titolo Testata:
EUROPEAN JOURNAL OF BIOCHEMISTRY
fascicolo: 19, volume: 268, anno: 2001,
pagine: 5037 - 5044
SICI:
0014-2956(200110)268:19<5037:CEPACO>2.0.ZU;2-C
Fonte:
ISI
Lingua:
ENG
Soggetto:
LIPID ACYL HYDROLASE; TRANSGENIC TOBACCO PLANTS; POTATO-TUBER PROTEIN; GENES; A(2);
Keywords:
patatin cloning; phospholipase A; active site dyad; characterization;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
18
Recensione:
Indirizzi per estratti:
Indirizzo: Egmond, MR Univ Utrecht, Dept Enzymol & Prot Engn, CBLE, Padualaan 8, NL-3584 CH Utrecht, Netherlands Univ Utrecht Padualaan 8 Utrecht Netherlands NL-3584 CH rlands
Citazione:
H.J.H.B. Hirschberg et al., "Cloning, expression, purification and characterization of patatin, a novelphospholipase A", EUR J BIOCH, 268(19), 2001, pp. 5037-5044

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 butwith an N-terminal histidine-tag. This patatin was overexpressed under thecontrol of the lac promotor in Escherichia coli strain DH5 alpha. 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 patatinpurified 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 (diC(8)PCho) (127 U.mg(-1)) and 1,2-dinonanoyl-sn-glycero-3-phosphocholine (diC(9)PCho) (109 U.mg(-1)). Recently, the structure of human cytosolic PLA(2) (cPLA(2)) 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.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 02/04/20 alle ore 12:39:51