Production of human skeletal alpha-actin proteins by the baculovirus expression system.
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.
Anthony Akkari
P
P
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.
Nowak
Kristen J
KJ
Beckman
Kaye
K
Walker
Kendall R
KR
Schachat
Fred
F
Laing
Nigel G
NG
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003850:Deoxyribonuclease I / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D018482:Muscle, Skeletal / Q000502:physiology* / Q000503:physiopathology; D009154:Mutation; D011108:Polymers / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000199:Actins; D011108:Polymers; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D003850:Deoxyribonuclease I