Expression of the antibody 14D9 in Nicotiana tabacum hairy roots
Keywords: catalytic antibodies, in vitro cultures, molecular farming, recombinant antibodies, tobacco.
Present address: #Fundación Pablo Cassará, Saladillo 2452, Ciudad de Buenos Aires, (C1144OFFX), Tel: 54 11 4886 5242, Fax: 54 11 4065.
Transgenic plants, those that integrate a foreign fragment of DNA in its own genome, are an economical alternative for large-scale production of recombinant proteins for industrial and pharmaceutical uses. Research in the past decades has revolutionized the use of therapeutically valuable proteins in a variety of clinical treatments and diagnosis. Most of the production has been performed in whole plant; however, not many proteins can be produced in plants at high levels. Anyway a 0.1-1.0% total soluble protein (TSP) yield, the typical level obtained for the production of pharmaceutical proteins, is competitive enough in order to make plants economically viable. Besides, protein production in plant cells is safer than traditional techniques (hybridomes and bacterial cell cultures) because of the lack of contamination with strange viral or bacterial materials, mammalian pathogens and other animal cell-culture contaminants. In vitro plant cell cultures, appeared as an interesting alternative when a foreign protein has to be synthesized under controlled environmental conditions or when the product is required in a quicker way than that obtained by agriculture procedure (Hellwig et al. 2004). In addition, another important advantage of in vitro culture is the possibility to recover the foreign protein from the culture medium which reduces the cost of down stream processing. Taking into account all the aspects cited before, protein production costs could be reduced by as much as 1000-fold compared with traditional production practices (Peterson and Arntzen, 2004). On the other hand, as the economic feasibility of plant-based systems depends on the concentration of foreign protein accumulated, preventing the degradation or loss of product after its assembly and/or secretion therefore it is of vital importance. It is also of importance the expression of the foreign protein in a cellular compartment where the degradation were diminished. The fusion of a protein to the C-terminal KDEL sequence for retention in and/or retrieve to the endoplasmic reticulum (ER) has been found to increase protein stability and to avoid addition of complex glycans in plant cells (Ko et al. 2003; Gomord et al. 2004). Also, the use of protein-stabilizing agents such as polyvinylpyrrolidone (PVP) and gelatin is one of the most promising methods for retaining foreign proteins secreted into plant culture medium. If the protein remains attached to biomass, permeabilising agents such as dimethylsulfoxide (DMSO) could be used (Wongsamuth and Doran, 1997; Magnuson, 1996). At present, in vitro cultures and particularly hairy roots have been recognized to be an alternative for foreign protein production. Hairy roots are a stable reproducible system which grows faster than suspension cell cultures and also they are able to grow in plant growth regulator-free culture medium. Considering the proteins that have been produced in plants, perhaps the most promising ones are monoclonal antibodies and antibody fragments. However, there have been few reports on antibody production by hairy root cultures (Wongsamuth and Doran, 1997; Sharp and Doran, 2001). The aim of this work is to study the expression of the 14D9 antibody in Nicotiana tabacum hairy root cultures. 14D9 is a murine antibody IgG1 type which catalyzes the stereoselective transformation of achiralenol ethers having a practical appeal for organic synthesis (Reymond et al. 1993; Zheng et al. 2003).
N. tabacum hairy roots were established at a frequency of above 85%. Among the different clones obtained which present differences regarding growth as well as in antibody expression levels. Clones Ac2 and AcK6 were selected for their characteristics of growth (high rate) and their capacity to express 14D9 antibody in a stable way. Hairy roots from line AcK6 grew faster and stronger than the wild type and line Ac2. That phenomenon is also observed in the whole plants and suspension cell cultures derived from AcK6 line. It could be attributed to the site of T-DNA integration in that transgenic line.
On the other hand the antibody yield (expressed as µg g-1FW) in line AcK6 was 3.21-fold higher than in line Ac2. The values obtained after 20 days of culture were 0.386 % TSP for clone Ac2 and 9.34 %TSP for clone AcK6. These results allow sustaining that preventing the secretion of protein by using the KDEL signal peptide for retention in the ER minimizes antibody fragmentation and improves antibody accumulation in plant cells (Tsoi and Doran, 2002). Also proteases in the secretory pathway and in the apoplast are avoided (Sharp and Doran, 2001). In the culture medium the highest values of antibodies were about 1% of the amounts reached in the biomass for both clones.
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