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 subsequently anion- and cation-exchange chromatography. All three purified proteins showed a single band of the expected molecular mass of 16 kDa in SDS-PAGE 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 previously prepared chicken and ovine leptins and to commercially available human leptin. The amino acid content of the purified proteins closely resembled the predicted composition. The biological activity of bovine leptin, porcine leptin, and porcine leptin analogue W4R/R5K was evidenced by their ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin receptor. All three proteins, as well as ovine and chicken leptins, but not human leptin, exhibited a very high degree of cross-immunoreactivity against antiserum raised against ovine leptin in rabbits. In contrast, none or very low cross-immunoreactivity was observed against antiserum raised against ovine leptin in goats.
Raver
N
N
Institute of Biochemistry, Food Science and Nutrition, The Hebrew University, Rehovot, Israel.
Gussakovsky
E E
EE
Keisler
D H
DH
Krishna
R
R
Mistry
J
J
Gertler
A
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002645:Chickens; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006041:Goats; D006801:Humans; D007106:Immune Sera; D020738:Leptin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine
D007106:Immune Sera; D020738:Leptin; D011994:Recombinant Proteins