THE ISOLATION OF NOVEL LEPTIN VARIANTS USING PHAGE DISPLAY

Leptin is a polypeptide hormone with important roles in a number of physiological pathways. Most significantly leptin acts a major regulator of energy metabolism and defects in hormone signalling form the basis of a number of human conditions, including morbid obesity, cardiovascular disease, and diabetes. Leptin is also an important immunomodulatory hormone and may contribute to a range of inflammatory diseases. For these reasons there is significant interest in the development of novel forms of leptin as possible therapeutic agents. Research into therapeutic applications of leptin has focused on attempts to engineer the leptin molecule to introduce properties such as improved affinity for the corresponding receptor, increased protein stability, and the development of both agonists and antagonists. These efforts have been largely based on rational engineering of the hormone based on structural studies and site-directed mutagenesis. Phage display of proteins and guided enrichment by selective biopanning is a powerful technique that allows the sampling of very large populations of protein variants.
In this study I expressed functional leptin on the surface of filamentous phage and used this technique to synthesise a library of random mutant leptins in the form of a phage library. A selective procedure was developed based on immobilised leptin receptor and a competitive binding strategy to select novel variants from the leptin mutant library with increased receptor affinity. The mutants were characterised and leptin proteins expressed and purified. The recombinant leptins were analysed for receptor stimulating activity and receptor affinity determined in real-time studies using biointerferometry. A novel leptin mutant was recovered with increased receptor affinity and agonist activity. This study established an approach for further phage based studies of leptin and other polypeptide hormones.