High yield expression and purification of human endothelin-1

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Big endothelin (Big ET) and endothelin (1-21) are biologically active peptides with significant physiological roles.
• Efficient and scalable production of these peptides is crucial for research and therapeutic development.

Purpose of the Study:

• To develop a robust method for the recombinant production and purification of human big endothelin (Big ET) and its active form, endothelin (1-21).
• To optimize a two-step purification strategy involving affinity chromatography and RP-HPLC.

Main Methods:

• Construction of a DNA construct encoding a fusion protein of maltose binding protein (MBP) and human Big ET, with a Factor Xa protease recognition site.
• Expression of the MBP-Big ET fusion product in DH5-alpha cells.
• Enzymatic cleavage of the fusion protein using trypsin and alpha-chymotrypsin.
• Purification of Big ET (1-37) using affinity chromatography with a specific peptide ligand.
• Final purification of endothelin (1-21) by reversed-phase high-performance liquid chromatography (RP-HPLC).

Main Results:

• High expression levels of the MBP-Big ET fusion product (approx. 100 µg/ml, 50% of total protein).
• Efficient cleavage of the fusion protein with approximately 70% yield for Big ET (1-37).
• Successful purification of Big ET (1-37) to 80% purity via affinity chromatography.
• Obtained endothelin (1-21) with 95% purity, yielding 3 µg per ml of bacterial broth.

Conclusions:

• The developed method enables efficient expression and purification of recombinant Big ET and endothelin.
• The two-step purification protocol provides high yields and purity of endothelin (1-21).
• This approach offers a scalable and effective strategy for producing biologically active endothelin peptides.