Pendergast A M, Clark R, Kawasaki E S, McCormick F P, Witte O N
Department of Microbiology and Molecular Biology Institute, University of California, Los Angeles 90024.
Oncogene. 1989 Jun;4(6):759-66.
The chronic myelogenous leukemia-associated P210 BCR-ABL oncogene protein product has been produced using the baculovirus expression system. High-level expression of the P210 BCR-ABL protein required the removal of GC rich 5' non-coding sequences. P210 BCR-ABL synthesized in insect cells is an active tyrosine protein kinase indistinguishable from P210 BCR-ABL isolated from human cells. Both proteins utilize angiotensin II as a phosphate acceptor in vitro with a Km for ATP of approximately 1.5 microM. P210 BCR-ABL produced in insect cells undergoes autophosphorylation in vitro and in vivo. Gel filtration of P210 BCR-ABL reveals that the protein elutes as a high molecular weight complex of about 800 kD. Approximately 4 to 5 mg of P210 BCR-ABL is produced in one liter of infected insect cells. Following cell disruption and a three-step ion exchange and gel filtration purification procedure, 0.4 mg of soluble P210 BCR-ABL is obtained per liter of suspension culture. An alternative procedure employing detergent extraction and immunoaffinity chromatography gave higher yields and purity from smaller amounts of infected cell extracts. The availability of intact, soluble and enzymatically active P210 BCR-ABL represents a significant advance for studying the biochemical and biophysical properties of the ABL oncogene family of proteins.
慢性粒细胞白血病相关的P210 BCR-ABL癌基因蛋白产物已通过杆状病毒表达系统生产出来。P210 BCR-ABL蛋白的高水平表达需要去除富含GC的5'非编码序列。在昆虫细胞中合成的P210 BCR-ABL是一种活性酪氨酸蛋白激酶，与从人细胞中分离出的P210 BCR-ABL无法区分。这两种蛋白在体外均以血管紧张素II作为磷酸受体，ATP的Km约为1.5微摩尔。在昆虫细胞中产生的P210 BCR-ABL在体外和体内都会发生自磷酸化。对P210 BCR-ABL进行凝胶过滤显示，该蛋白以约800 kD的高分子量复合物形式洗脱。在一升受感染的昆虫细胞中可产生约4至5毫克的P210 BCR-ABL。经过细胞破碎以及三步离子交换和凝胶过滤纯化程序后，每升悬浮培养物可获得0.4毫克可溶性P210 BCR-ABL。另一种采用去污剂提取和免疫亲和色谱的方法，从较少量的受感染细胞提取物中获得了更高的产量和纯度。完整、可溶且具有酶活性的P210 BCR-ABL的可得性代表了在研究ABL癌基因家族蛋白的生化和生物物理特性方面取得的重大进展。
