Production of human skeletal α-actin proteins by the baculovirus expression system
Production of human skeletal α-actin proteins by the baculovirus expression system
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DOI：
10.1016/s0006-291x(03)01133-1
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发表时间：
2003-07-18
影响因子：
3.1
通讯作者：
Laing, NG
中科院分区：
文献类型：
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作者：
Akkari, PA;Nowak, KJ;Laing, NG
人类骨骼肌α-肌动蛋白基因内的突变导致三种不同的骨骼肌疾病。突变蛋白的功能研究是必要的，以更好地了解这些疾病的发病机制，然而，没有令人满意的系统表达的突变肌肉肌动蛋白蛋白已可用。我们研究了杆状病毒表达载体系统（BEVS）的丰富生产正常和突变的骨骼肌α-肌动蛋白。我们表明，在BEVS中产生的非突变肌动蛋白的行为类似于天然肌动蛋白，如DNase I亲和纯化，蛋白质印迹，和连续的聚合和解聚循环所示。此外，我们证明了突变肌动蛋白在BEVS中的产生，而不会损害表达它们的昆虫细胞。因此，BEVS是研究导致人类疾病的突变肌动蛋白的首选方法。(C)2003 Elsevier Science（美国）。All rights reserved.
Mutations within the human skeletal muscle alpha-actin gene cause three different skeletal muscle diseases. Functional studies of the mutant proteins are necessary to better understand the pathogenesis of these diseases, however, no satisfactory system for the expression of mutant muscle actin proteins has been available. We investigated the baculovirus expression vector system (BEVS) for the abundant production of both normal and mutant skeletal muscle alpha-actin. We show that non-mutated actin produced in the BEVS behaves similarly to native actin, as shown by DNase I affinity purification, Western blotting, and consecutive cycles of polymerisation and depolymerisation. Additionally, we demonstrate the production of mutant actin proteins in the BEVS, without detriment to the insect cells in which they are expressed. The BEVS therefore is the method of choice for studying mutant actin proteins causing human diseases. (C) 2003 Elsevier Science (USA). All rights reserved.