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.