Clostridium botulinum C3 ADP-ribosyltransferase gene. Cloning, sequencing, and expression of a functional protein in Escherichia coli.
C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho and rac proteins in eukaryotic cells. The enzyme was purified from a culture filtrate of C. botulinum type C strain 003-9, and the amino acid sequence from the amino-terminal Ser to Asn192 was determined by Edman degradation. Using a set of degenerate primers based on the sequence, we amplified a part of the gene for this enzyme by polymerase chain reaction. A 2.1-kilobase pair HincII fragment of C. botulinum DNA containing the whole structural gene was then identified by Southern analysis with the polymerase chain reaction product as a probe, and the complete nucleotide structure of the gene together with flanking regions was determined by cloning and DNA sequencing the HincII fragment. The gene encodes a protein of 244 amino acids with a Mr of 27,362 which begins with a putative signal peptide of 40 amino acids. Escherichia coli carrying this gene produced the active enzyme, and about 60% of it was found in the culture medium. Immunoblot analysis with antiserum against the enzyme revealed the presence of two immunoreactive proteins of 27 and 23 kDa in the cytoplasmic/membrane fraction and only the 23-kDa protein in the periplasm and the medium, suggesting that the enzyme expressed is processed in the E. coli, exported into the periplasm and released into the culture medium.
Nemoto
Y
Y
Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
Namba
T
T
Kozaki
S
S
Narumiya
S
S
D036002:ADP Ribose Transferases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D001905:Botulinum Toxins; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003014:Clostridium botulinum / Q000201:enzymology* / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000235:genetics; D015183:Restriction Mapping; D016415:Sequence Alignment
D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins