Method for increasing the secretion of recombinant proteins

#The present invention relates to the optimization of recombinant plant or fungal expression systems with the aid of suitable expression methods.

#The production of recombinant proteins in a concentration range of ng/l to μg/l in plants has already been described in a number of publications. The publication WO 01/25456 A2 relates to a method for the production of proteins that are present in moss without lysing the moss tissues or moss cells and in addition mentions that the productivity may be increased by the use of PVP in a culture medium. By way of example, this is shown for the recombinant production of VEGF121, a small glycosylated homodimer with a size of 28 kDa (Baur et al., Journal of Biotechnology 119 (2005): 332-342). Baur et al. describe the transient transformation of moss using PEG. The medium employed for the production of VEGF was free of PVP or PEG.

#Drake et al., Plant Mol. Biol. 52 (2003): 233-241, describe the recombinant expression of antibodies in a culture of tobacco plant tissue, i.e. of root tissue. The antibodies were secreted by a process of rhizosecretion in the microgram (μg) range. Effective plant expression systems based on rhizosecretion are mostly hairy root cultures that can be stimulated by infecting any type of plant with Agrobacterium rhizogenes (Gaume et al., Plant Cell Rep. 21 (2003): 1188-1193).

#The present invention is further illustrated by the following Figures and Examples, without being limited to these specific embodiments of the present invention, however.

#FIG. 1 shows the synergistic effects of an increase in osmolarity by the addition of NaCl and the polymer PEG on the secretion of a recombinantly produced antibody;

#FIG. 2 shows additional production-increasing effects of mineral substances for stimulating plant growth (Ca nitrate);

#The present invention provides a method that enables the continuous secretion of recombinant proteins from plant cultures into the culture medium. In said method, the producing plants are submerged in a liquid culture and are photoautotrophically cultivated in specific mineral media. The culture media are optimized with respect to an optimal nutritional maintenance of moss plants, but do not per se cause the secretion of recombinantly produced larger proteins into the medium, which proteins first accumulate in the apoplast and can be measured as “intracellular” following the disruption of the moss tissue.

#The apoplastic restraint of the proteins can be reversed by the addition of osmotically active substances (“secretion components”), such as NaCl and polyethylene glycol (PEG) 4000, to the culture medium. This leads to a quick release of the accumulated proteins into the medium and in the further course of the culture to a continuous and temporally immediate secretion of newly synthesized proteins.

#500 ml Erlenmeyer flasks were filled with 180 ml of sterile Knop or WM01 medium, adjusted to pH 4.5 to 6 (2-(N-morpholino) ethanesulfonic acid buffer, “MES”) and inoculated with a freshly turraxed suspension culture of Physcomitrella protonema from a recombinant strain produced by a test protein. The inoculation density was 0.1 g dry weight/l. The cultures were sealed in a sterile and gas-permeable manner and cultivated in an atmosphere supplemented with 2% CO2. Upon growth to a cell density of 1 to 3 g/l, the secretion components were added in the form of a sterile concentrate. During the cultivation procedure, the parameters of culture density (g dry weight/l), intracellular and extracellular IgG titer (mg/l) were determined at regular intervals.