Protease activity in the medium of larch (<em>Larix</em> spec.) embryogenic suspension cultures and medium-protein stabilization by compatible solutes

Abstract

The stability of recombinant, secreted protein in the medium of transgenic plant cell cultures heavily determines the resulting protein yield, which is a crucial factor for every production system. In order to gain more knowledge about the feasability of rapidly growing larch (Larix sp.) embrogenic cell cultures as a possible expression system for recombinant proteins, spent cell culture medium was characterized in this study. An accumulation of endogenous proteins could be observed in the medium of larch embrogenic suspension cultures which reached up to 1750 μg per g fresh weight. In contrast, low protease activity accumulated within a typical 14-day culture period in the medium. This activity was up to 20 times lower than the protease activity in two callus-derived suspension cultures of tobacco (genotype R1 and BY-2) which were measured in parallel. To asses the stability of foreign proteins, medium aliquots were spiked with Immunoglobulin G (IgG) and the amount of protein degradation was determined after 23 h of incubation by SDS-PAGE. The loss of IgG was comparable in three different larch genotypes, resulting in a mean loss of 18 % during the incubation time. This loss could remarkably be diminished by the addition of ectoin derivatives, known to be protein-protective „compatible solutes“ of bacterial origin. The most effective one was hydroxyectoin which resulted in a 76 % reduction of the observed IgG degradation. The stabilization of proteins in plant cell culture medium by compatible solutes is shown here for the first time. The possible mechanism of the stabilizing effect is discussed.