Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

NICOTIANA TABACUM AS BIOFACTORY FOR TRANSGLUTAMINASE ENZYME

MARINIELLO L.*, SORRENTINO A.*, PAOLINO M.*, TORTIGLIONE C.**, PORTA R.*

* Department of Food Science, University of Naples “Federico II”, Parco Gussone 80055 Portici, Naples, Italy

loremari@unina.it

** Research Institute for Vegetable and Flower Breeding, CNR, Portici, Naples, Italy

Recombinant proteins from plants is one of the most exciting and fastest developing areas in biology. In fact, plants have considerable potential for the production of therapeutic and industrial biomolecules (Giddings et al., 2000). Our research is addressed to utilize Nicotiana tabacum as a cheap source of an enzyme which could useful for both pharmaceutical and industrial fields. Such is transglutaminase (TGase, E.C. 2.3.2.13), an enzyme capable of catalyzing acyl-transfer reactions introducing covalent cross-links between proteins as well as peptides and various primary amines. When the e- amino groups of lysine residues act as acyl acceptors, e-(g-glutamyl)-lysine bonds are formed both intra- and inter-molecularly. For the type of reaction catalyzed, TGase results a useful tool to cross-link proteins of different origin. Substrate specificity is related to the existence of different isoforms of TGase, which constitute a family of enzyme widely distributed in Mammals, but also identified in Invertebrates and Plants. Among well known mammalian TGases is Factor XIII, an heterotetrameric form present in human plasma where stabilizes the fibrin clot during the final stage of blood-clotting sequence (Sobel & Gawinowicz, 1996). Keratinocyte TGase present in skin is involved in the terminal differentiation of keratinocytes in cross-linking of a number of structural proteins (Thacher & Rice, 1985). Prostate TGase is responsible for the clotting of rodent seminal plasma (Folk, 1980). Tissue TGase is the most widespread member of the family with several hypothesized roles including an involvement in apoptosis (Fesus et al., 1987), in stabilization of the extracellular matrix (Aeschlmann et al., 1995) and in transmembrane signal mediation, acting as a G-protein (Gah) (Im et al., 1997). It is well known that, among the others mammalian members of the TGase family, tissue TGase is able to cross-link proteins of different origin, including food proteins (as caseins, soy proteins, and gluten), besides structural proteins (as actin, fibronectin and others). In this study we have subcloned human tissue TGase cDNA in PKYLX71, a vector useful for the expression of foreign proteins in plants, downstream the constitutive CaMV 35S2 promoter. Nicotiana tabacum plants have been transformed via Agrobacterium tumefaciens, following the resistance to kanamicine, whose gene is harbored in PKYLX71 vector. The presence of the exogenous gene has been verified by PCR using TGase cDNA specific primers. To date, transgenic plants have been analyzed by Northern and Western blot analyses.

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