Nobre, LígiaSandner, PeterBarbas, AnaBotnaru, Adriana2020-01-162022-10-312019-12-132019http://hdl.handle.net/10362/91278The protein A, the main receptor in mammalian cells for the compound X, is a heterodimeric hemoprotein composed of an a and b subunits, that catalyzes the conversion of a substrate into the a product. Four different subunits have been identified, namely, a1 a2 b1 and b2; while the a1/b1 and a2/b1 heterodimeric isoforms have been extensively characterized and their function well described, the role of b2 subunit has remained elusive. The aim of this study is the expression and purification of the b2 subunit, and its functional characterization. To this end, recombinant baculovirus encoding the b2 subunit were generated and used to infect Sf9 insect cells. To optimize the expression of b2 several small scale tests were performed, and the best condition was identified and used to produce and purify of the enzyme via an affinity and a size exclusion chromatography. The purified b2 protein is catalytically active in the presence of two different co-factors and was identified in a monomeric form. However, results also suggest the presence of a homodimeric state. The b2 subunit lacks the spectroscopic features of a hemic protein and does not respond to compound X, indicating that the protein does not bind the heme group, in opposition to other reported b2. Moreover, the protein is not activated by compound Y, which is a well know protein A activator. In addition, co-expression tests of b2 subunit with a2 showed that a2/b2 complex does not seem to be active, unlike the a2/b1 heterodimeric isoform, which is a catalytically active and compound X-sensitive enzyme, as previously reported. Altogether, the work presented in this master thesis suggests that b2 subunit is a catalytically active enzyme, lacking the heterodimeric structure usually reported to A proteins.engmaster thesis