Subcloning, expression, purification, and characterization of recombinant human leptin-binding domain.
A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.
Sandowski
Yael
Y
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University, Rehovot 76100, Israel.
Raver
Nina
N
Gussakovsky
Eugene E
EE
Shochat
Suzan
S
Dym
Orly
O
Livnah
Oded
O
Rubinstein
Menachem
M
Krishna
Radha
R
Gertler
Arieh
A
D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D002645:Chickens; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D020738:Leptin / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D054411:Receptors, Leptin; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012756:Sheep; D020349:Surface Plasmon Resonance
D017931:DNA Primers; C515448:LEPR protein, human; D020738:Leptin; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011994:Recombinant Proteins