Expression and Purification of Soluble STAT5b/STAT3 Proteins for SH2 Domain Binding Assay

Abstract

When a large hydrophobic full-length protein is expressed in bacteria, it is often challenging to obtain recombinant proteins in the soluble fraction. One way to overcome this challenge is expression of deletion mutants that have improved solubility while maintaining biological activity. In this chapter, we describe a protocol for expression of truncated forms of STAT5b and STAT3 proteins that are soluble and retain SH2-mediated activity for phospho-Tyr peptide recognition.

Towards the Inhibition of Protein–Protein Interactions (PPIs) in STAT3: Insights into a New Class of Benzothiadiazole Derivatives

“…The pTyr peptide probes used in this study were 5-carboxyfluorescein (FITC)-GpYLPQTV for STAT3 and (FITC)-GpYDKPHVL for STAT1. Plasmid construction and protein expression were performed as previously described [ 52 ]; the experimental method is briefly summarized as follows. Considering that the expression of the full-length protein is troublesome, a truncated form of STAT3 (136-705) was constructed and chromatographically purified (AKTA FPLC system; GE Healthcare Bio-Sciences, Piscataway, NJ, USA).…”