EXPRESSION AND PURIFICATION OF THE CANINE 54-KDA SUBUNIT OF SIGNAL RECOGNITION PARTICLE AS A HIS-TAGGED PROTEIN FROM ESCHERICHIA-COLI

The 54-kDa subunit of the signal recognition particle (SRP) binds nasc ent secretory polypeptides, binds the 7SL RNA (SRP RNA) component of S RP, and hydrolyzes GTP. Limited proteolysis of SRP 54-kDa suggests the protein has two domains, termed the G (G;TP-binding) and M (methionin e-rich) domains. The M domain is predicted to contain a number of amph iphilic helices, which provide a binding cleft for signal sequences. I n order to obtain sufficient material for studies of relationships bet ween structure and function, we have expressed the canine cDNA encodin g the 54-kDa subunit in Escherichia coli using a T7 expression system. To aid purification, the protein was expressed with an amino-terminal extension encoding an initiating methionine and 10 histidine residues followed by an enterokinase cleavage site; 0.3mg of HIS:SRP 54-kDa wa s purified to give a single band on SDS-PAGE in 20% yield from 500 mi of cultured E. coli. Purified HIS:SRP 54-kDa was shown to be folded in to the G and M domains, to inhibit the translocation of pre-prolactin into canine microsomes, and to bind mammalian SRP RNA only in the pres ence of the 19-kDa subunit of SRP. (C) 1996 Academic Press, Inc.