Expression of recombinant growth factors using plant-based biorreactors ? a comparative approach

MÜLLER, CAROLINA; MIRKIN, FEDERICO G.; PEREZ CASTRO, CAROLINA; WIRTH, SONIA A.; SEGRETIN, MARÍA EUGENIA

Ciudad de Córdoba

Congreso; XXXII Reunión Argentina 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 biorreactors. 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 (hFGFb). 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 transgen 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 hFGFb 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.