Hsu C Y, Mohammadi M, Nathan M, Honegger A, Ullrich A, Schlessinger J, Hurwitz D R
Rorer Biotechnology, Inc., King of Prussia, Pennsylvania 19406.
Cell Growth Differ. 1990 Apr;1(4):191-200.
We have generated a recombinant baculovirus using the high expression vector pVL941 containing the complementary DNA encoding the intracellular domain of the human epidermal growth factor receptor (EGFR-IC). Upon infection of Spodoptera frugiperda insect cells, protein tyrosine kinase-active EGFR-IC was produced. The expressed protein has a molecular weight of 61,000 and is specifically recognized by antibodies directed against peptides representing different regions of human EGFR-IC. Upon sonication of infected cells, EGFR-IC was detected in both the soluble and insoluble fractions of the cell lysate. About 20-50% of the expressed EGFR-IC was soluble. Metabolic labeling and protein analyses showed that EGFR-IC comprised 7% of newly synthesized proteins in the cytoplasmic lysate and 0.1-0.2% of the total soluble protein. We have used a three-step purification procedure (fast-Q-Sepharose and phenyl-Sepharose column chromatographies and 30% ammonium sulfate precipitation) to purify EGFR-IC to 85% purity with 15-20% recovery from the initial soluble lysate. A yield of 3-4 mg of purified EGFR-IC has been consistently produced from 20 roller bottles with 2-4 x 10(8) infected cells/bottle. The tyrosine kinase activity was retained through purification. The enzyme demonstrated much higher autophosphorylation activity in the presence of Mn2+ than Mg2+. Phosphopeptide mapping revealed the same autophosphorylation sites utilized by EGFR-IC as those identified in wild-type EGFR. EGFR-IC-catalyzed phosphorylation of either a synthetic peptide representing the major autophosphorylation site or angiotensin II showed that the baculovirus-expressed EGFR-IC exhibits similar enzymatic kinetic characteristics to the intact activated EGFR kinase.
我们使用了含有编码人表皮生长因子受体（EGFR-IC）胞内结构域互补DNA的高表达载体pVL941，构建了一种重组杆状病毒。感染草地贪夜蛾昆虫细胞后，产生了具有蛋白酪氨酸激酶活性的EGFR-IC。所表达的蛋白分子量为61,000，能被针对代表人类EGFR-IC不同区域的肽段的抗体特异性识别。对感染细胞进行超声处理后，在细胞裂解物的可溶性和不溶性部分均检测到了EGFR-IC。所表达的EGFR-IC约20 - 50%是可溶的。代谢标记和蛋白质分析表明，EGFR-IC在细胞质裂解物中新合成的蛋白质中占7%，在总可溶性蛋白质中占0.1 - 0.2%。我们采用三步纯化程序（快速Q - 琼脂糖凝胶柱层析、苯基 - 琼脂糖凝胶柱层析和30%硫酸铵沉淀），从初始可溶性裂解物中纯化得到纯度为85%的EGFR-IC，回收率为15 - 20%。从20个滚瓶中，每个滚瓶接种2 - 4×10⁸个感染细胞，一直能稳定产生3 - 4mg纯化的EGFR-IC。酪氨酸激酶活性在纯化过程中得以保留。该酶在存在Mn²⁺时比在Mg²⁺时表现出更高的自磷酸化活性。磷酸肽图谱分析显示，EGFR-IC的自磷酸化位点与野生型EGFR中确定的位点相同。EGFR-IC对代表主要自磷酸化位点的合成肽或血管紧张素II的催化磷酸化表明，杆状病毒表达的EGFR-IC与完整的活化EGFR激酶具有相似的酶动力学特征。
