Construction of pRSET-sfGFP Plasmid for Fusion-Protein Expression, Purification and Detection

Abstract

Green fluorescent protein (GFP) is widely used as an excellent expression tag for fusion proteins, which can improve their expression while preserving their function and native-like structure. Fusion protein method allows the purification and the detection of a protein of interest even when no specific antibody is available. This study aimed to design a system for protein expression and detection using a new super-folder derivative of GFP (sfGFP) as a fusion partner. This included the construction of the protein expression plasmid pRSET-sfGFP by cloning sfGFP gene downstream of the N-terminal 6×His tag in the T7 promoter-plasmid pRSET. The soluble expressed sfGFP protein (27 kDa) from this plasmid in the cytoplasm of E. coli was purified using metal affinity chromatography, as shown after SDS-PAGE separation and blue gel staining. In order to detect sfGFP, as single or in fusion proteins, in ELISA and immunoblotting analysis, sfGFP-specific polyclonal antibodies were produced in rabbit after immunization with three injections with the pure sfGFP prepared in Freund’s adjuvant. Two-step antibody purification, using protein A-Sepharose and sfGFP-coupled Sepharose affinity chromatography columns, was performed to obtain highly reactive and pure sfGFP-specific IgG. This system will provide an efficient tool for the expression, purification and detection of many proteins, having problems in solubility and stability, as fusion partners with sfGFP.