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

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 2.14

Plant expression of a potent antimicrobial scFv recombinant antibody

Donini M.*, Pashkoulov D.*, Gerioni M.*, Polonelli L.**

*) ENEA-Casaccia, UTS Biotecnologie, Protezione della Salute e degli Ecosistemi Roma

**) Università di Parma, Sezione di Microbiologia-Dipartimento di Patologia e Medicina di Laboratorio

transient expression, PVX, antimicrobial, scFv

Plants represent a valuable tool for the production of peptides and proteins mostly due to the ease of plant transformation, to the generally high expression yields and to low production costs. There are many examples of field trials for large scale production of high value added molecules such as vaccines, biopharmaceuticals and antibodies for therapy and diagnosis (Giddings, G. et al., 2000 Nature 18: 1151-1155).

In this work we report about the transient expression in plant of a recombinant antibody (single chain Fv fragment, named scFvH6) with a potent microbicidal activity, that has been already characterised for its in vitro toxic activity against animal prokaryotic and eukaryotic pathogens and showing a significant therapeutical activity in a rat candidiasis in vivo model (Magliani, W. et al. 1997, Nat. Biotechnology 15: 155-158; Beninati C., et al., 2000 Nat. Biotechnology 18: 1060-1064), has been transiently expressed in plants. Transient scFv expression has been obtained using a recombinant plant virus (Potato Virus X) infection system well known in literature for the high expression yields of recombinant proteins. The antimicrobial scFvH6 was cloned in the pPVX121 vector harbouring a secretory plant signal derived from the polygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris. Nicotiana benthamiana and N. tabacum plants were infected with the recombinant PVX and tested by RT-PCR for scFv expression. Western blot analysis showed that very low amounts of soluble scFv were found in the intercellular fluids and in total protein soluble plant extracts from the infected plants (about 0,005% of total soluble protein content). Fractionated protein extraction from N. benthamiana symptomatic leaves revealed high accumulation of the scFvH6 in non-soluble membranous protein fractions associated to nuclei, chloroplasts, mitochondria and ER membranes. This work demonstrates that scFvH6 tends to aggregate to cell membranes when directed to the secretory pathway, and therefore plants are not suitable for the production of high protein amounts at least in a soluble form. Purification of the scFv from the insoluble protein extracts can be still be envisaged. Notably the stable expression such antimicrobial scFv antibody in plant may also represent an effective strategy to protect plant from pathogens.