Bacterial expression and purification of biologically active human TFF2

Area of Science:

• Biochemistry
• Molecular Biology
• Gastroenterology

Background:

• Human trefoil factor 2 (hTFF2) is crucial for gastrointestinal mucosal healing, promoting cell migration and inhibiting apoptosis.
• Obtaining native hTFF2 from human sources is challenging, and existing recombinant methods often yield fusion proteins.
• There is a need for a reliable method to produce active, non-fused hTFF2 for research and therapeutic development.

Purpose of the Study:

• To develop a method for producing native human trefoil factor 2 (hTFF2) with preserved biological activity.
• To express and purify recombinant hTFF2 in Escherichia coli.
• To confirm the biological functions of the produced recombinant hTFF2 in vitro.

Main Methods:

• The hTFF2 open reading frame was cloned into a pET-32a(+) vector for expression in E. coli as an hTFF2-TRX fusion protein.
• Purification involved Nickel-NTA affinity chromatography and reverse-phase HPLC.
• The fusion protein was cleaved using Factor Xa to release native hTFF2, followed by further purification and identification via SDS-PAGE and Western blotting.

Main Results:

• Recombinant hTFF2-TRX fusion protein was successfully expressed and purified to >95% purity.
• Cleavage with Factor Xa yielded native hTFF2 at approximately 5 mg/L.
• The purified recombinant hTFF2 demonstrated biological activity by promoting IEC-6 cell migration and wound healing (via ERK1/2 activation) and inhibiting HCT-116 cell apoptosis.

Conclusions:

• A high-yield method for producing biologically active, native recombinant hTFF2 in E. coli was established.
• The recombinant hTFF2 effectively mimics the known functions of native hTFF2 in promoting mucosal healing.
• This recombinant hTFF2 is a valuable tool for studying hTFF2's molecular mechanisms and for developing hTFF2-based therapeutics.