Preparation of recombinant bovine, porcine, and porcine W4R/R5K leptins and comparison of their activity and immunoreactivity with ovine, chicken, and human leptins

Recombinant ovine Ala-leptin (GenBank Accession No. U84247, of ovine leptin ), previously prepared in our laboratory in prokaryotic expression plasmid pMON3401, was mutated using a mutagenesis kit to prepare plasmids encoding for bovine (GenBank Accession No. U50365) and porcine (GenBank Accession No . U59894) leptins and for porcine leptin analogue W4R/R5K. Escherichia coli cells transformed with these plasmids overexpressed large amounts of these proteins upon induction with nalidixic acid. The expressed proteins, found in inclusion bodies, were refolded and purified to homogeneity using subse quently anion- and cation-exchange chromatography, All three purified prote ins showed a single band of the expected molecular mass of 16 kDa in SDS-PA GE in the presence of reducing agent and were composed of 90-100% monomers, Proper refolding was evidenced by comparing their CD spectra to those of p reviously prepared chicken and ovine leptins and to commercially available human leptin, The amino acid content of the purified proteins closely resem bled the predicted composition. The biological activity of bovine leptin, p orcine leptin, and porcine leptin analogue W4R/R5K was evidenced by their a bility to stimulate proliferation of leptin-sensitive BAF/3 cells transfect ed with a long form of human leptin receptor. All three proteins, as well a s ovine and chicken leptins, but not human leptin, exhibited a very high de gree of cross-immunoreactivity against antiserum raised against ovine lepti n in rabbits. In contrast, none or very low cross-immunoreactivity was obse rved against antiserum raised against ovine leptin in goats, (C) 2000 Acade mic Press.