Anthony Akkari P, Nowak Kristen J, Beckman Kaye, Walker Kendall R, Schachat Fred, Laing Nigel G
Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Australian Neuromuscular Research Institute, 4th Floor, 'A' Block, QEII Medical Centre, Nedlands, 6009, Western Australia, Australia.
Biochem Biophys Res Commun. 2003 Jul 18;307(1):74-9. doi: 10.1016/s0006-291x(03)01133-1.
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.
人类骨骼肌α-肌动蛋白基因内的突变会引发三种不同的骨骼肌疾病。对突变蛋白进行功能研究对于更好地理解这些疾病的发病机制很有必要，然而，目前尚无用于表达突变型肌肉肌动蛋白的理想系统。我们研究了杆状病毒表达载体系统（BEVS），以大量生产正常和突变型骨骼肌α-肌动蛋白。我们发现，杆状病毒表达载体系统中产生的非突变型肌动蛋白的行为与天然肌动蛋白相似，这通过脱氧核糖核酸酶I亲和纯化、蛋白质免疫印迹以及连续的聚合和解聚循环得以证明。此外，我们还证明了杆状病毒表达载体系统能够产生突变型肌动蛋白，且不会对其表达所在的昆虫细胞造成损害。因此，杆状病毒表达载体系统是研究导致人类疾病的突变型肌动蛋白的首选方法。
