Rapid Production of Recombinant Human SLFN14 Ribonuclease and Stoichiometric Analysis by Mass Photometry

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Ribonucleases are vital RNA-binding proteins involved in gene regulation.
• Many human ribonucleases are poorly understood due to production challenges.
• Human Schlafen 14 (SLFN14) ribonuclease function and structure require further elucidation.

Purpose of the Study:

• To establish an efficient method for recombinant human SLFN14 production and characterization.
• To investigate the oligomeric state and RNA binding of SLFN14.
• To provide a reliable protein source for downstream structural and biochemical analyses.

Main Methods:

• Transient expression of Flag-tagged SLFN14 in HEK293 cells.
• Two-step purification: anti-Flag affinity chromatography and size exclusion chromatography.
• Mass photometry for single-molecule analysis of protein-RNA interactions.

Main Results:

• A rapid and efficient workflow for SLFN14 purification was established.
• SLFN14 exists as a stable homodimer bound to cellular RNA under physiological conditions.
• High salt concentrations induce RNA release but maintain the homodimer structure.

Conclusions:

• The developed method enables production of functional recombinant human SLFN14.
• SLFN14's homodimeric state and RNA-binding properties were characterized.
• This approach is applicable to other mammalian ribonucleases and RNA-binding proteins.