Barley cell culture as a producer of heterologous protein:Dissertation

The purpose of the present study was to demonstrate the possibility of using the stable cell culture of barley (Hordeum vulgare var. Pokko) established in our laboratory, in the production of heterologous proteins. Production of a cellulolytic enzyme, heat stable endoglucanase EGI from the filamentous fungi Trichoderma reesei, was chosen as an example. Bombardment of DNA into intact barley cells was used for gene transfer. Two genes were simulta neously transferred, one coding for a selectable marker, antibiotic resistance, and one for the enzyme to be studied. The transferred cultures were cloned by sub- culturing a small cell cluster of 80-120 cells five times on selection medium containing the antibiotic geneticin. When assayed for ß-glucanase activity after four months, 80 % of the clones showed integration of both heterologous genes in the plant genome. One clone produced 20 times the average amount of EGI and was chosen for further studies. The production of EGI was followed over 25 weeks. The productivity appeared to be stable indicating that the cell line was a pure clone and that the foreign gene was stable in the genome. Barley cell cultures were successfully preserved in liquid nitrogen. Regrowth started rapidly after thawing and in two weeks a new suspension culture could be started. No change in growth behaviour or in productivity was observed. The gene egll containing a fungal signal sequence caused secretion of the heterologous protein, EGI. Accumulation of EGI commenced in the medium from the start of the cultivation and in the stationary phase a 25 fold higher activity was found in the medium than in the cells which was regarded as proof for secretion. The production of heterologous EGI affected the growth of the suspension culture. The cell mass yield was somewhat lower than in the untransformed cell culture. During the growth of barley cells in suspension, a colloidal precipitate appeared in the medium. The precipitate could be removed from the EGI preparations by gel filtration and anion-exchange chromatography on DEAE cellulose. The heterologous EGI from barley showed considerable similarity to the fungal EGI of T. reesei. The molecular weight of the heterologous extracellular barley EGI (54-55 kD) was slightly lower than the corresponding value of fungal EGI (55-58 kD). Isoelectric focusing and activity blotting thereof with agar plates containing barley ß-glucan also revealed two EGI active bands at pI values of 4.40 and 4.45 for the heterologous barley EGI and 4.75 and 4.80 for the native EGI. The substrate specificity of hetero logous EGI was in accordance with that of the fungal EGI. Barley ß-glucan was the preferred substrate and high xylanolytic activity was also observed. The beneficial effect of both fungal and heterologous barley EGI was demonstrated in laboratory scale mashing experiments. It was possible to remove the residual phenoxyherbicide 2,4-dichlorophenoxyacetic acid used as growth promoting factor in plant cell cultures, from the EGI preparations by gel filtration. The present study serves as an example on the use of stable plant cell culture in production of heterolo gous proteins