Abstract
The Ser/Arg-rich splicing factors (SR proteins) constitute a crucial protein family in alternative splicing, comprising twelve members characterized by unique repetitive Arg-Ser dipeptide sequences (RS) and one to two RNA-recognition motifs (RRM). The RS regions of SR proteins undergo variable phosphorylation, resulting in unphosphorylated, partially phosphorylated, or hyper-phosphorylated states based on functional requirements. Despite the identification of the SR protein family over 30 years ago, the purification of native SR proteins in soluble form at large quantities has presented challenges due to their low solubility. This protocol delineates a method for acquiring soluble, full-length, unphosphorylated, hypo- and hyper-phosphorylated SRSF1, a prototypical SR family member. Notably, this protocol facilitates the purification of SRSF1 in ample quantities suitable for NMR, as well as various biophysical and biochemical studies. The methodologies and principles outlined herein are expected to extend beyond SRSF1 protein production and can be adapted for purifying other SR protein family members or SR-related proteins, such as snRNP70 and U2AF-35. Given the involvement of these proteins in numerous essential biological processes, this protocol will prove beneficial to researchers in related fields. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Purification of SRSF1 from E. coli Support Protocol: Purification of ULP1 Basic Protocol 2: Purification of hypo-phosphorylated SRSF1 from E. coli Basic Protocol 3: Purification of hyper-phosphorylated SRSF1 from E. coli.