TY - JOUR
T1 - Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach
AU - Lackmann, Martin
AU - Bucci, Tamara
AU - Mann, Richard J.
AU - Kravets, Lucy A.
AU - Viney, Elizabeth
AU - Smith, Fiona
AU - Moritz, Robert L.
AU - Carter, Wendy
AU - Simpson, Richard J.
AU - Nicola, Nicos A.
AU - Mackwell, Karen
AU - Nice, Edouard C.
AU - Wilks, Andrew F.
AU - Boyd, Andrew W.
PY - 1996/3/19
Y1 - 1996/3/19
N2 - 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 × 106-fold purification of an ≈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.
AB - 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 × 106-fold purification of an ≈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.
UR - http://www.scopus.com/inward/record.url?scp=0029981272&partnerID=8YFLogxK
U2 - 10.1073/pnas.93.6.2523
DO - 10.1073/pnas.93.6.2523
M3 - Article
C2 - 8637907
AN - SCOPUS:0029981272
SN - 0027-8424
VL - 93
SP - 2523
EP - 2527
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 6
ER -