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首页> 美国卫生研究院文献>Proceedings of the National Academy of Sciences of the United States of America >Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach.
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Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach.

机译：使用基于生物传感器的亲和力检测方法纯化EPH样受体HEK的配体。

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摘要

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.
机译：筛选技术的发展仅允许通过DNA或蛋白质序列比较来鉴定生长因子受体，因此需要分离相应配体生长因子的新颖方法。 EPH样受体酪氨酸激酶（RTK）HEK（人EPH样激酶）先前被鉴定为LK63人pre-B细胞系上的膜抗原，在白血病标本和细胞系中过表达提示其在肿瘤发生中的作用。我们开发了一种基于生物传感器的方法，使用固定的HEK受体外域来检测和监测HEK配体的纯化。一种蛋白质纯化方案（包括HEK亲和色谱法）从人胎盘条件培养基中获得了约23 kDa蛋白质的1.8 X 10（6）倍纯化。在最初和最终纯化步骤中对特定sHEK（可溶性HEK胞外域）配体相互作用的分析表明，原料中配体的浓度为40 pM，Kd为2-3 nM。由于纯化的配体在N端被封闭，我们生成了胰蛋白酶肽，S-吡啶基乙基化蛋白的7个胰蛋白酶片段的N端氨基酸序列分析明确匹配了AL-1的序列，AL-1是最近报道的有关EPH-就像RTK REK7（Winslow，JW，Moran，P.，Valverde，J.，Shih，A.，Yuan，JQ，Wong，SC，Tsai，SP，Goddard，A.，Henzel，WJ，Hefti，F.，Beck ，KD＆Caras，IW（1995）Neuron 14，973-981）。我们的发现证明了生物传感器技术在配体纯化中的应用，并且表明，与EPH-like RTK家族的其他配体一样，AL-1与一个以上的受体结合。

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