Expression and purification of the canine 54-kDa subunit of signal recognition particle as a his-tagged protein fromescherichia coli

Patel, Sarju and Austen, Brian M. 1996. Expression and purification of the canine 54-kDa subunit of signal recognition particle as a his-tagged protein fromescherichia coli. Protein Expression and Purification 8 (3) , pp. 283-294. 10.1006/prep.1996.0102

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Abstract

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 inEscherichia coliusing 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 culturedE. 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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine
Publisher:	Elsevier
ISSN:	1046-5928
Last Modified:	31 Jan 2025 17:15
URI:	https://orca.cardiff.ac.uk/id/eprint/175610

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