title: Expression, purification and refolding of active durum wheat (Triticum durum Desf.) secretory phospholipase A2 from inclusion bodies of Escherichia coli.
abstract: Recently, a durum wheat (Triticum durum Desf.) secretory phospholipase A2 (TdsPLA2III) was identified in leaves as potentially involved in plant responses to conditions of limiting water supply. Therefore, to allow future functional studies on TdsPLA2III and shed further light on the involvement of sPLA2 isoforms in specific plant functions, here we report a protocol for the overexpression of TdsPLA2III in Escherichia coli in the form of inclusion bodies, and for its purification and refolding. The use of the Gateway system (Invitrogen) allows the expression of a large quantity of the mature form (without the signal peptide) of TdsPLA2III with an N-terminal 6×His-tag, for purification using Ni-affinity chromatography. The purified recombinant 6×His-TdsPLA2III fusion protein is then refolded using a step-wise dialysis approach. About 40mg purified and active protein was obtained from 1L of cell culture. This recombinant 6×His-TdsPLA2III protein shows PLA2 activity, as it can hydrolyze linoleate from the sn-2 position of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine. Moreover, it has some features that are typical of other known plant sPLA2s: Ca(2+)-dependence, inhibition by the disulfide bond reducing agent dithiothreitol, and resistance to high temperature.
authors.affiliation: Consiglio per la Ricerca e la sperimentazione in Agricoltura, Centro di Ricerca per la Cerealicoltura, S.S. 673, Km 25,200, 71122 Foggia, Italy. Electronic address: angeloverlotta@yahoo.it.
authors.forename: Angelo
authors.initials: A
authors.lastname: Verlotta
authors.affiliation: Consiglio per la Ricerca e la sperimentazione in Agricoltura, Centro di Ricerca per la Cerealicoltura, S.S. 673, Km 25,200, 71122 Foggia, Italy. Electronic address: daniela.trono@entecra.it.
authors.forename: Daniela
authors.initials: D
authors.lastname: Trono
mesh_terms: D000595:Amino Acid Sequence; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006358:Hot Temperature; D002479:Inclusion Bodies / Q000201:enzymology*; D019787:Linoleic Acid / Q000096:biosynthesis; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000737:chemistry; D064801:Phospholipase A2 Inhibitors / Q000494:pharmacology; D054497:Phospholipases A2, Secretory / Q000096:biosynthesis / Q000235:genetics*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014908:Triticum / Q000201:enzymology* / Q000235:genetics
chemical_list: D010713:Phosphatidylcholines; D064801:Phospholipase A2 Inhibitors; D011993:Recombinant Fusion Proteins; C055402:1-palmitoyl-2-linoleoylphosphatidylcholine; D019787:Linoleic Acid; D054497:Phospholipases A2, Secretory; D004229:Dithiothreitol
keywords: Escherichia coli; Inclusion bodies; Phospholipase A(2); Recombinant protein; Refolding