The Drosophila behaviour/human splicing (DBHS) proteins are a family of RNA binding proteins which mediate an array of functions relating to transcription regulation, transcriptional and post-transcriptional processing and nuclear export1. They also bind to the long non-coding RNA (lncRNA) NEAT1 to form membrane-less subnuclear bodies called paraspeckles which may mediate gene expression through the sequestering of RNA2. In the clinical context, DBHS proteins are implicated in neuronal development, innate immunity and cancer1. As the functions of these proteins are predominantly mediated through RNA binding, knowledge of their RNA binding specificity and structural information on paraspeckles will provide crucial information on how these proteins function and their role in mediating physiological and pathological processes.
In characterising the RNA binding specificity of DBHS proteins, we express various wild type DBHS protein constructs and measure their binding affinity with various RNA binding partners using microscale thermophoresis (MST). We then express mutant versions of these proteins where key residues in their RNA recognition motifs (RRMs) are mutated and compare their binding affinity with their wild type counterparts. In the expression of these proteins, we encountered difficulties in purifying proteins at sufficiently high concentration to conduct the MST experiments. Here we detail our attempts at increasing the yield of purified proteins through influencing their stability through buffer optimization.