Paraspeckles are nuclear membraneless organelles. They consist of long non-coding RNA (lncRNA) and various RNA-binding proteins such as splicing factor proline and glutamine rich (SFPQ) and Non-POU domain containing octamer binding protein (NONO). They form around the lncRNA NEAT1 (nuclear paraspeckle assembly transcript 1). Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA-binding protein located in the nucleus and is one of the key proteins in paraspeckle formation. An increased abundance of these nuclear organelles in neurons is characteristic of amyotrophic lateral sclerosis (ALS). The role and formation of paraspeckles in the context of ALS is not yet known. In this work, we focused on the preparation of the paraspeckle protein hnRNPK to study paraspeckles in association with ALS. To this end, we incorporated the HNRNPK and HNRNPK-NeonGreen sequences into a bacterial expression vector pMCSG7 using Gibson assembly. We successfully expressed both HNRNPK constructs in the E. coli strain BL21[DE3] and determined the optimal conditions for expression that can be used for larger-scale production of HNRNPK. For further research, it is necessary to carry out the expression process on a larger scale and carry out purification procedures for individual protein constructs.
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