Extraction and purification of immunoglobulin G with aqueous biphasic systems

The immune system is able to produce antibodies, which have the capacity to recognize and to bind to foreign molecules or pathogenic organisms. Currently, there are a diversity of diseases that can be treated with antibodies, like immunoglobulins G (IgG). Thereby, the development of cost-efficient processes for their extraction and purification is an area of main interest in biotechnology. Aqueous biphasic systems (ABS) have been investigated for this purpose, once they allow the reduction of costs and the number of steps involved in the process, when compared with conventional methods. Nevertheless, typical ABS have not showed to be selective, resulting in low purification factors and yields. In this context, the addition of ionic liquids (ILs) as adjuvants can be a viable and potential alternative to tailor the selectivity of these systems. In this work, ABS composed of polyethylene glycol (PEG) of different molecular weight, and a biodegradable salt (potassium citrate) using ILs as adjuvants (5 wt%), were studied for the extraction and purification of IgG from a rabbit source. Initially, it was tested the extraction time, the effect on the molecular weight of PEG in a buffer solution of K3C6H5O7/C6H8O7 at pH≈7, and the effect of pH (59) on the yield (YIgG) and extraction efficiency (EEIgG%) of IgG. The best results regarding EEIgG% were achieved with a centrifugation step at 1000 rpm, during 10 min, in order to promote the separation of phases followed by 120 min of equilibrium. This procedure was then applied to the remaining experiments. The results obtained in the study of PEGs with different molecular weights, revealed a high affinity of IgG for the PEG-rich phase, and particularly for PEGs of lower molecular weight (EEIgG% of 96 % with PEG 400). On the other hand, the variation of pH in the buffer solution did not show a significant effect on the EEIgG%. Finally, it was evaluated the influence of the addition of different ILs (5% wt) on the IgG extraction in ABS composed of PEG 400 at pH≈7. In these studies, it was possible to obtain EEIgG% of 100% with the ILs composed of the anions [TOS]-, [CH3CO2]-and Cl-, although the obtained YIgG% were lower than 40%. On the other hand, the ILs composed of the anions Br-, as well as of the cation [C10mim]+, although not leading to EEIgG% of 100%, provide an increase in the YIgG%. ABS composed of PEG, a biodegradable organic salt and ILs as adjuvants, revealed to be an alternative and promising method to purify IgG. However, additional studies are still required in order to reduce the loss of IgG.

Molecular regulation of cork development: the QsMYB1 gene

Cork is a natural and renewable material obtained as a sustainable product from cork oak (Quercus suber L.) during the tree’s life. Cork formation is a secondary growth derived process resulting from the activity of cork cambium. However, despite its economic importance, only very limited knowledge is available about the molecular mechanisms underlying the regulation of cork biosynthesis and differentiation. The work of this PhD thesis was focused on the characterization of an R2R3-MYB transcription factor, the QsMYB1, previously identified as being putatively involved in the regulatory network of cork development. The first chapter introduces cork oak and secondary growth, with special emphasis on cork biosynthesis. Some findings concerning transcriptional regulation of secondary growth are also described. The MYB superfamily and the R2R3-MYB family (in particular) of transcription factors are introduced. Chapter II presents the complete QsMYB1 gene structure with the identification of two alternative splicing variants. Moreover, the results of QsMYB1 expression analysis, done by real-time PCR, in several organs and tissues of cork oak are also reported. Chapter III is dedicate to study the influence of abiotic stresses (drought and high temperature) and recovery on QsMYB1 expression levels. The effects of exogenous application of phytohormones on the expression profile of QsMYB1 gene are evaluated on Chapter IV. Chapter V describes the reverse genetic approach to obtain transgenic lines of Populus tremula L. x tremulóides Michx. overexpressing the QsMYB1 gene. Finally, in Chapter VI the final conclusions of this PhD thesis are presented and some future research directions are pointed based on the obtained results.