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 nascent secretory polypeptides, binds the 7SL RNA (SRP RNA) component of SRP, and hydrolyzes GTP. Limited proteolysis of SRP 54-kDa suggests the protein has two domains, termed the G (GTP-binding) and M (methionine-rich) domains. The M domain is predicted to contain a number of amphiphilic helices, which provide a binding cleft for signal sequences. In order to obtain sufficient material for studies of relationships between structure and function, we have expressed the canine cDNA encoding 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 was purified to give a single band on SDS-PAGE in 20% yield from 500 ml of cultured E. coli. Purified HIS-SRP 54-kDa was shown to be folded into the G and M domains, to inhibit the translocation of pre-prolactin into canine microsomes, and to bind mammalian SRP RNA only in the presence of the 19-kDa subunit of SRP.
Patel
S
S
Department of Surgery, St. George's Hospital Medical School, London, United Kingdom.
Austen
B M
BM
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D011388:Prolactin / Q000037:antagonists & inhibitors / Q000235:genetics; D011498:Protein Precursors / Q000037:antagonists & inhibitors / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D018271:Signal Recognition Particle / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D011498:Protein Precursors; D011994:Recombinant Proteins; D018271:Signal Recognition Particle; D006639:Histidine; C017671:preprolactin; D011388:Prolactin