Construction of pRSET-sFGFP plasmid for fusion-protein expression, purification and detection

Abstract EN

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 (sGFP) as a fusion partner.

This included the construction of the protein expression plasmid pRSET- sGFP by cloning sGFP gene downstream of the N-terminal 6xHis tag in the T7 promoter-plasmid pRSET.

The soluble expressed sGFP 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 sGFP, as single or in fusion proteins, in ELISA and immunoblotting analysis, sGFP-specific polyclonal antibodies were produced in rabbit after immunization with three injections with the pure sGFP prepared in Freund's adjuvant.

Two-step antibody purification, using protein A-Sepharose and sGFP-coupled Sepharose affinity chromatography columns, was performed to obtain highly reactive and pure sGFP-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 sGFP.