Clostridium botulinum C3 ADP-ribosyltransferase gene. Cloning, sequencing, and expression of a functional protein in Escherichia coli.

摘要: Abstract C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho rac proteins in eukaryotic cells. The enzyme was purified from a culture filtrate C. type strain 003-9, the amino acid sequence amino-terminal Ser to Asn192 determined Edman degradation. Using set degenerate primers based on sequence, we amplified part gene for this polymerase chain reaction. A 2.1-kilobase pair HincII fragment DNA containing whole structural then identified Southern analysis with reaction product as probe, complete nucleotide structure together flanking regions cloning sequencing fragment. encodes protein 244 acids Mr 27,362 begins putative signal peptide 40 acids. Escherichia coli carrying active enzyme, about 60% it found medium. Immunoblot antiserum against revealed presence two immunoreactive 27 23 kDa cytoplasmic/membrane fraction only 23-kDa periplasm medium, suggesting that expressed processed E. coli, exported into released