Expression, partial purification and functional properties of the muscle-specific calpain isoform p94

The muscle-specific calpain isoform p94 has high propensity to autocatalyti c degradation, thus no significant amounts of the intact active protein hav e been available so far. As a result, aspects like its regulation (via Ca2 and other factors) and its intracellular localization are unknown or obscu re. In this work, large amounts of human p94 have been produced in insect c ells using a recombinant baculovirus expression system. Although most of th e protease was recovered in an insoluble and catalytically inactive form, t he soluble fraction contained amounts of intact active p94 adequate for its characterization. His-tagged recombinant p94, obtained by the same express ion system, was partially purified as an active product. Both the unmodifie d and the partially purified His-tagged p94 bound calcium with high affinit y, and their autolytic activity required Ca2+. The sensitivity of the catal ytic activity of the recombinant protease to Ca2+ was very high. In fact, p 94 in soluble cell extracts autolysed to a significant extent even in the p resence of submicromolar Ca2+ levels. Thus, in analogy to what demonstrated for the ubiquitous m- and mu-calpain isoforms, intracellular Ca2+ might be one of the factors controlling the activity of this muscle-specific calpai n isoform.