Open AccessIsolation andin vitro, expression of the ricin A-chain gene: effect of deletions on biological activity
Author(s) -
Anders Sundan,
Grethe Evensen,
E Hornes,
Anita Mathiesen
Publication year - 1989
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/17.4.1717
Subject(s) - biology , ricin , microbiology and biotechnology , gene , messenger rna , biochemistry , start codon , mutant , stop codon , gene expression , expression vector , recombinant dna , toxin
The gene encoding the ricin A-chain was isolated and subcloned into an in vitro expression vector downstream from the SP6 promotor. mRNA encoding the A-chain strongly inhibited the translational activity of reticulocyte lysates. The inhibition correlated with glycosylase activity on rRNA, and could be abolished by addition of antibodies specific for ricin. mRNA generated after linearization of the vector at unique restriction sites within the A-chain coding sequence did not inhibit, except after linearization with ScaI. Also mutants lacking the 28 N-terminal amino acids of native A-chain strongly inhibited the lysates. However, in both cases no glycosylase activity could be observed. We also show that the lack of a stop codon in mRNA does not affect the level of expression as assayed here.