High-level production of functional muscle alpha-tropomyosin in Pichia pastoris

Although numerous studies have reported the production of skeletal muscle a lpha -tropomyosin in E. coli, the protein needs to be modified at the amino terminus in order to be active. Without these modifications the protein do es not bind to actin, does not exhibit head-to-tail polymerization, and doe s not inhibit the actomyosin Mg2+-ATPase in the absence of troponin. On the other hand, the protein produced in insect cells using baculovirus as an e xpression vector (Urbancikova, M., and Hitchcock-DeGregori, S. E., J. Biol. Chem., 269, 24310-24315, 1994) is only partially acetylated at its amino t erminal and therefore is not totally functional. In an attempt to produce a n unmodified functional recombinant muscle alpha -tropomyosin for structure -function correlation studies we have expressed the chicken skeletal alpha -tropomyosin cDNA in the yeast Pichia pastoris. Recombinant protein was pro duced at a high level (20 mg/L) and was similar to the wild type muscle pro tein in its ability to polymerize, to bind to actin and to regulate the act omyosin S1 Mg2+-ATPase. (C) 2001 Academic Press.