MOLECULAR-CLONING AND BACTERIAL EXPRESSION OF CDNA FOR RAT CALPAIN-II80 KDA SUBUNIT

The complete cDNA of 3.2 kb for rat calpain II large subunit has been constructed from library- and polymerase chain reaction-derived fragme nts, and sequenced. The cDNA encodes a protein of 700 amino acids havi ng 93% sequence identity with human calpain II, and 61% identity with human calpain I. The gene possesses 21 exons, of which exons 3-21 have been mapped over 33 kb of the rat genome. A new phagemid expression v ector was created from pT7-7 by insertion of the f1 origin and mutatio n of an NdeI to an NcoI site. Rat calpain II cDNA ligated into this ve ctor expressed in Escherichia coli an 80 kDa protein identical in size to highly purified rat calpain II; this protein was specifically reco gnized on immunoblots by an affinity-purified anti-rat calpain II anti body. This is the second mammalian calpain II large subunit to be full y sequenced, and the first to be artificially expressed.