Ribonucleases are a specialized class of RNA binding proteins that catalyze RNA cleavage and contribute to diverse biological processes including gene regulation. Nevertheless, the structure, function, and regulation of many human ribonucleases remain poorly characterized due to the challenges of recombinant protein production. This method describes an efficient approach for the transient expression, purification, and stoichiometric analysis of the human Schlafen 14 (SLFN14) ribonuclease. Recombinant Flag-tagged SLFN14 was expressed in HEK293 suspension cells and isolated using a rapid two-step purification workflow consisting of anti-Flag affinity chromatography followed by low-resolution size exclusion chromatography. Mass photometry, a sensitive single-molecule technique, revealed that in physiological salt conditions SLFN14 forms a stable homodimer bound to cellular RNA, whereas high salt promotes RNA dissociation while preserving the homodimer arrangement. The resulting recombinant human SLFN14 protein is suitable for downstream structural studies and qualitative biochemical assays. This rapid approach for recombinant human protein isolation and sample characterization is broadly applicable for the production of other mammalian ribonucleases and RNA binding proteins intended for biophysical and biochemical characterization.
Masuga et al. (Fri,) studied this question.