Expression, Purification and Characterization of an Enzyme Isoform Involved in Cyclic Nucleotide Signaling in Humans

Abstract

The soluble guanylyl cyclase (sGC), the main nitric oxide (NO) receptor in mammalian cells, is a heterodimeric hemoprotein composed of an α and β subunits, that catalyzes the conversion of guanosine-5’-triphosphate (GTP) into the second messenger 3’,5’-cyclic guanosine monophosphate (cGMP). Four different subunits have been identified, namely, α₁, α₂, β₁ and β₂; while the α₁/β₁ and α₂/β₁ heterodimeric isoforms have been extensively characterized and their function well described, the role of β₂ subunit has remained elusive. The aim of this study is the expression and purification of the human β₂ subunit, and its functional characterization. To this end, recombinant baculovirus encoding the human β₂ subunit were generated and used to infect Sf9 insect cells. To optimize the expression of β₂several small scale tests were performed, and the best expression condition was identified and used to produce and purify the enzyme via an affinity and a size exclusion chromatography.

The purified human β₂ protein is catalytically active in the presence of 3 mM Mg²⁺ and 3 mM Mn²⁺ and was identified in a monomeric form. However, results also suggest the presence of a homodimeric state. The human β₂ subunit lacks the spectroscopic features of a hemic protein and does not respond to NO, indicating that the protein does not bind the heme group, in opposition to the reported to rat β₂. Moreover, the protein is not activated by BAY 60-2770, which is a well know hemefree-sGC activator. In addition, co-expression tests of β₂ subunit with α₂ showed that α2/β2 complex does not seem to be active, unlike the α₂/β₁heterodimeric isoform, which is a catalytically active and NOsensitive enzyme, as previously reported.

The work presented in this master thesis suggests that human β₂subunit is a catalytically active enzyme, lacking the heterodimeric structure usually reported to sGC proteins.