Engineering and use of (32)P-labeled human recombinant interleukin-11 for receptor binding studies.
Human interleukin-11 (hIL-11) is a pleiotropic cytokine that is involved in numerous biological activities such as hematopoiesis, osteoclastogenesis, neurogenesis and female fertility. IL-11 is obviously a key reagent to study the IL-11 receptors. However, conventional radio-iodination techniques lead to a loss of IL-11 bioactivity. Here, we report the construction and the production of a new recombinant human IL-11 (FP Delta IL-11). In this molecule, a specific phosphorylation site (RRASVA) has been introduced at the N-terminus of rhIL-11. It can be specifically phosphorylated by bovine heart protein kinase and accordingly, easily radiolabeled with (32)P. A high radiological specific activity (250,000 c.p.m x ng(-1) of protein) was obtained with the retention of full biological activity of the protein. The binding of (32)P-labeled FP Delta IL-11 to Ba/F3 cells stably transfected with plasmids encoding human IL-11 receptors alpha and beta chains (IL-11R alpha and gp130) was specific and saturable with a high affinity as determined from Scatchard plot analysis. Availability of this new ligand should prompt further studies on IL-11R structure, expression and regulation.
Wang
Xiao-Ming
XM
Institut Pasteur de Bruxelles, Belgium.
Wilkin
Jean-Marc
JM
Boisteau
Olivier
O
Harmegnies
Dimitri
D
Blanc
Chrystel
C
Vandenbussche
Paul
P
Montero-Julian
Félix A
FA
Jacques
Yannick
Y
Content
Jean
J
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002478:Cells, Cultured; D006801:Humans; D017370:Interleukin-11 / Q000378:metabolism*; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D015202:Protein Engineering; D018121:Receptors, Cytokine / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017370:Interleukin-11; D010761:Phosphorus Radioisotopes; D018121:Receptors, Cytokine; D011994:Recombinant Proteins