TOBACCO IN VITRO CULTURES AS BIOFACTORIES OF THE CATALYTIC ANTIBODY 14D9

LÓPEZ, J.; LENCINA, F.; CAPELLO, M.; PARMA, Y.; NELSON., G; MARCONI, P.L.; ALVAREZ, M.A.

Ciudad de México, México

Congreso; First International Congress on Biotechnology and Bioengineering. International Initiatives for Sustainable Development.; 2008

In vitro plant cell cultures are an environmental friendly alternative for large-scale production of recombinant proteins for industrial and pharmaceutical uses. They do not have contamination with bacterial endotoxins, mammalian pathogens and other animal cell-culture contaminants which makes them a safer production system than the traditional ones. Besides, in this way the foreign protein could be synthesized faster and under more controlled environmental conditions than in crops. Also, it is possible to recover the foreign protein from the culture medium reducing the cost of down-stream processing. Several strategies are used for improving the performance of such cultures. One of them is the fusion of the protein to the C-terminal KDEL sequence for retention in and/or retrieve to the endoplasmic reticulum (ER) that has been found to increase protein stability and to avoid addition of complex glycans in plant cells. On the other hand, the use of protein-stabilizing agents such as polyvinylipirrolidone (PVP) and gelatin is one of the most promising methods for increasing foreign protein yields. If the protein remains attached to the biomass, permeabilising agents such as dimethylsulfoxide (DMSO) could also be used. The aim of this work was to study the expression of the 14D9 antibody in hairy roots and suspension cell cultures of Nicotiana tabacum. 14D9 is a murine antibody IgG1- type which catalyzes the stereoselective transformation of achiral enol ethers having a practical application for organic synthesis. Particularly, we evaluated the influence on cell growth and antibody production of the KDEL ER- retention signal and of PVP (1.0, 1.5 and 2.0 g l-1), gelatin (1.0, 5.0 and 9-0 g l-1) and DMSO (2.8 % and 5.0 % V/V). We have demonstrated the ability of N. tabacum hairy roots and suspension cell cultures to express the functional antibody. As for hairy root cultures, the clone harboring the KDEL-sequence (Ab-KDEL) have grown faster (μ= 0.190 d-1) than both the wild type (μ = 0.105 d-1) and the line expressing the secretory version of the antibody (sec-Ab) (μ= 0.060 d-1). The highest antibody yield has been 5.95 ± 0.38 μgml-1for clone sec-Ab and 20.82 ± 0.25 μgml-1for clone Ab-KDEL. In cell suspension cultures the highest µ has been observed in the Ab-KDEL clone (0.149 d-1). The Ab-KDEL cell suspension cultures have shown approximately 5 -fold higher 14D9 yield than the sec-Ab line but it has been remarkably lower than the level attained in hairy roots. Also, we have been able to establish that the addition of PVP, gelatin and DMSO has enhanced antibody accumulation in the biomass of both types of cultures.