Influence of the sequence KDEL on the expression of the 14D9 murine antibody by nicotiana tabacum hairy root cultures

MARTINEZ, C.; SILVANA PETRUCCELLI; GUILIETTI, A. M.; ALVAREZ, M. A.

Barcelona

Congreso; 7th International Congress of Plant Molecular Biology; 2003

Plant Molecular Biology Society

As a heterologous antibody expression system, hairy roots are capable of high levels of production of assembled antibodies (Wongsamuth & Doran, 1997). The aim of this work was to study the influence of a KDEL sequence on the production of antibodies by tobacco hairy roots. Hairy roots cultures were initiated from transgenic tobacco plants expressing the murine antibody 14D9 with/without this ER retention sequence using Agrobacterium rhizogenes LBA 9402. Shake flasks containing 40 mL MSRT medium were each inoculated with 0.200 mg FW root tips from 16 day-old cultures of antibody-producing hairy roots. Triplicate flasks were harvested at intervals over a period of 30 days. Measurements of biomass (fresh weight) and antibody accumulation levels (by ELISA) in the roots and medium culture were carried out. Antibody assembly and integrity were also examined by Western blotting. The line which carries the sequence KDEL showed a faster (u= 0.19 d-1) growth respect to the other line (u=0.09 d-1) and the wild type (u=0.1 d-1). Antibody accumulation level (0.1 mg 14D9/gFW) and the final biomass (2.3gFW mL-1) were also higher in that hairy root clone. No antibody accumulation nor protease activity were detected on the culture medium. We demonstrated the ability of N.tabacum hairy roots to express the 14D9 antibody, and the positive influence of the sequence KDEL on the antibody expression.