Purification and characterization of a novel cell-penetrating carrier similar to cholera toxin chimeric protein

•A novel cell-penetrating carrier similar to cholera toxin chimeric protein was purified and characterized.•The protein complex was successfully obtained using an in vitro recombination method.•It bound more strongly to monosialoganglioside (GM1) than (CTB)5 at low concentrations.•The transmembrane function of TAT in this protein complex was also maintained.•The expression vector provides a feasible expression frame for constructing several new macromolecular protein drugs.

Developing a recombinant vector for noninvasively delivering biological macromolecules into the brain is important. This study constructed and purified a protein complex based on the cholera toxin (CT) molecular structure. Enhanced green fluorescent protein (EGFP)-modified A2 subunits of CT (CTA2) were used as tracer molecules for introduction of transactivator of transcription (TAT) through the A subunit into cells. The protein complex EGFP-CTA2-TAT/(CTB)5 (CTB: B subunit of CT) was obtained using an in vitro recombination method and verified by monosialoganglioside-enzyme-linked immunosorbent assay and high performance liquid chromatography assay. The protein complexes bound more strongly to monosialoganglioside (GM1) than (CTB)5 at low concentrations (0.625–1.25 μg/mL). In vitro assays revealed that the transmembrane function of TAT was also maintained. The GM1-binding activity and cell membrane-penetrating ability suggested that a CT structure-based protein complexes could be used to design a delivery carrier for intranasal administration through GM1 binding. The expression vector introduced in this study provides a feasible expression frame for constructing several new macromolecular protein drugs for effective cell penetration.