Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that primarily affects motor neurons and causes their degradation. They discovered 20 genes whose mutations are thought to be associated with the development of ALS. The most common mutation is in the C9ORF72 gene (chromosome 9 open reading frame 72), where there is an increase in intronic repeats of GGGGCC (G4C2)n, which causes sequestration of some RNA-binding proteins. Some of the mutations are also in the genes that encode paraspeckle proteins and cause their translocation from the nucleus to the cytoplasm, affecting paraspeckle’s functions. Paraspeckles are nuclear ribonucleoprotein bodies without a membrane, consisting of the non-coding RNA NEAT1 (Nuclear Paraspeckle Assembly Transcript 1) and its associated proteins, among which the key proteins are NONO (Non-POU Domain-Containing Octamer-Binding) and SFPQ (Splicing Factor Proline and Glutamine Rich). The mutations associated with ALS also lead to altered function of the NONO protein, an RNA- and DNA-binding protein that belongs to the DBHS (Drosophila Behavior/Human Splicing) protein family. They found that it binds to (G4C2)n-repeat transcripts together with SFPQ protein to form paraspeckle-like bodies, which may be one of the pathological mechanisms of ALS. To investigate the interactions within the paraspeckle-like bodies, we attempted to express the NONO protein in fusion with maltose-binding protein (MBP). We prepared two constructs by inserting the NONO coding sequence into the pMCSG7 vector with the MBP coding sequence at the N- and C- termini. When both variants were expressed in E. coli BL21[DE3] cells, the fusion proteins were first isolated by nickel affinity chromatography, with the variant with MBP at the N- terminus being isolated in higher quantity. We also attempted to isolate both fusion proteins using an MBP affinity column, but this was less efficient than isolation by nickel affinity chromatography. To isolate only the NONO protein, we cleaved the His-MBP tag from the fusion protein with MBP at the N- terminus with TEV protease and then attempted to purify it by size exclusion chromatography, but we had high losses. In addition, we found that neither of the fusion proteins was stable at 4 °C, as both fusion proteins aggregated or degraded. We need to optimize the isolation of the NONO protein to obtain enough of the protein for interaction analysis in paraspeckle-like bodies.
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