Biological activities of recombinant chicken leptin C4S analog compared with unmodified leptins

The chicken leptin sequence, in contrast to mammalian leptins, contains an unpaired Cys at position 3 of the original cDNA (AF012727). The presence of an extra Cys may confer a different structure and affect the leptin's biol ogical activity. To address this, we studied the effects of wild-type and m utated (C4S) chicken leptins in vitro and in vivo and compared them with ma mmalian leptin prepared from ovine leptin cDNA. The prokaryotic expression vector pMON, encoding full-size A(-1) chicken leptin (AF012727), was mutate d using a mutagenesis kit, yielding the C4S analog. Escherichia coli cells transformed with this vector overexpressed large amounts of chicken leptin C4S upon induction with nalidixic acid. The expressed protein, found in the inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharos e column, yielding three electrophoretically pure fractions, eluted from th e column by 100, 125, and 150 mM NaCl, respectively. All three fractions sh owed a single band of the expected molecular mass (16 kDa) and were compose d of >95% monomeric protein. Proper refolding was evidenced by comparing th e circular dichroism spectrum of the analog with spectra of nonmutated chic ken and ovine leptins. The biological activity of the C4S analog was eviden ced by its ability to stimulate proliferation of leptin-sensitive BAF/3 cel ls transfected with a long form of human leptin receptor construct similar to its nonmutated counterpart, indicating that Cys4 plays no role in leptin activity. The in vitro activity of both wild-type and mutated chicken lept ins was similar to 10-fold lower than that of ovine leptin. After intraveno us or intraperitoneal injections, C4S analog and the nonmutated chicken and ovine leptins all lowered the food intake of starved 9-day-old broiler or 5-wk-old layer male chickens by 11-34%. Monitoring food behavior revealed t hat the attenuated food intake resulted not from a decreased number of appr oaches to the feeders but from a decrease in the average time spent eating during each approach.