Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach.
Advances in screening technologies allowing the identification of growth factor receptors solely by virtue of DNA or protein sequence comparison call for novel methods to isolate corresponding ligand growth factors. The EPH-like receptor tyrosine kinase (RTK) HEK (human EPH-like kinase) was identified previously as a membrane antigen on the LK63 human pre-B-cell line and overexpression in leukemic specimens and cell lines suggested a role in oncogenesis. We developed a biosensor-based approach using the immobilized HEK receptor exodomain to detect and monitor purification of the HEK ligand. A protein purification protocol, which included HEK affinity chromatography, achieved a 1.8 X 10(6)-fold purification of an approximately 23-kDa protein from human placental conditioned medium. Analysis of specific sHEK (soluble extracellular domain of HEK) ligand interactions in the first and final purification steps suggested a ligand concentration of 40 pM in the source material and a Kd of 2-3 nM. Since the purified ligand was N-terminally blocked, we generated tryptic peptides and N-terminal amino acid sequence analysis of 7 tryptic fragments of the S-pyridylethylated protein unequivocally matched the sequence for AL-1, a recently reported ligand for the related EPH-like RTK REK7 (Winslow, J.W., Moran, P., Valverde, J., Shih, A., Yuan, J.Q., Wong, S.C., Tsai, S.P., Goddard, A., Henzel, W.J., Hefti, F., Beck, K.D., & Caras, I.W. (1995) Neuron 14, 973-981). Our findings demonstrate the application of biosensor technology in ligand purification and show that AL-1, as has been found for other ligands of the EPH-like RTK family, binds more than one receptor.
Lackmann
M
M
Cooperative Research Centre for Cellular Growth Factors, Growth Regulation Laboratory, Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
Bucci
T
T
Mann
R J
RJ
Kravets
L A
LA
Viney
E
E
Smith
F
F
Moritz
R L
RL
Carter
W
W
Simpson
R J
RJ
Nicola
N A
NA
Mackwell
K
K
Nice
E C
EC
Wilks
A F
AF
Boyd
A W
AW
D000595:Amino Acid Sequence; D001483:Base Sequence; D015374:Biosensing Techniques; D002478:Cells, Cultured; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D036383:Ephrin-A2; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D011485:Protein Binding; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D036121:Receptor, EphA3; D017978:Receptors, Growth Factor / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification*
D036383:Ephrin-A2; D006133:Growth Substances; D008024:Ligands; D017978:Receptors, Growth Factor; D014157:Transcription Factors; D020794:Receptor Protein-Tyrosine Kinases; D036121:Receptor, EphA3; D004259:DNA-Directed DNA Polymerase