Expression of recombinant Growth Factors using plan-based bioreactors: a comparative approach.

MULLER, CAROLINA; BRAVO-ALMONACID, FERNANDO; PEREZ CASTRO, CAROLINA; MIRKIN, FEDERICO; SEGRETIN, MARIA EUGENIA; WIRTH, SONIA

Congreso; XXXII Encuentro Argentino de Fisiología Vegetal (RAFV) y XVI Congreso Latinoamericano de Fisiología Vegetal.; 2018

Growth factors are paracrine signaling proteins with mitotic and angiogenic activities which are also involved in the maintenance of the pluripotency state in Human Embryonic Stem Cells (hESC). Given the importance of hESCs in regenerative medicine and research, the aim of this work is to develop different strategies for the production of human recombinant growth factors (rGFs) using plant-based bioreactors. Chloroplast transformation allows the expression of high levels of recombinant proteins in plants and was therefore chosen as our first approach to produce human Fibroblast Growth Factor (FGF). We cloned a 6xhis-tagged version of hFGFb in a plastid transformation vector (pUTR-hFGFb) which was used to transform Nicotiana tabacum leaves by particle bombardment. PCR analysis of regenerated shoots confirmed the integration of the transgene in five independent transplantomic lines. These lines are being subjected to successive regeneration rounds to achieve homoplasmy. In addition, we are evaluating the production of FGF in Nicotiana benthamiana plants by agroinfiltration, a transient expression strategy. We adapted the pEAQ vector for rapid subcloning of recombinant proteins fused to different localization signals, to compare the accumulation of recombinant proteins in different subcellular compartments. Through the comparison of these strategies effectiveness, we attempt to optimize recombinant hFGFb production.