Cloning, nucleotide sequencing, and expression of tetanus toxin fragment C in Escherichia coli

Abstract

The amino acid sequence of the first 30 residues of fragment C of tetanus toxin was determined, and a mixture of 32 complementary oligonucleotides, each 17 bases long, was synthesized. A 2-kilobase (kb) EcoI fragment of Clostridium tetani DNA was identified by Southern blotting and was cloned into the Escherichia coli plasmid vector pAT153 with the 32P-labeled oligonucleotide mixture as a probe. A second 3.2-kb BglII fragment was identified and cloned with the 2-kb EcoRI fragment as a probe. The nucleotide sequence of 1.8 kb of this DNA was determined and was shown to encode the entire fragment C and a portion of fragment B of tetanus toxin. The tetanus DNA was expressed in E. coli with pWRL507, a plasmid vector containing the trp promoter and a portion of the trpE gene. The trpE-tetanus fusion proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were shown to react with anti-fragment C antibody.Tetanus toxin is a potent inhibitor of the central nervous system. It causes spastic paralysis by blocking the release of inhibitory transmitters from inhibitory synapses (for a review, see reference 1). The exact mode of action of tetanus toxin at the molecular level remains unknown. The toxin, produced by Clostridium tetani, is synthesized as a 150,000-dalton polypeptide (Fig. 1). Upon lysis of the bacterium, the toxin is released from the cells, concomitantly with proteolytic cleavage of the molecule by an endogenous protease. The resulting molecule, termed "extracellular toxin," is composed of two fragments designated the light and heavy chains (13). These chains are held together by one or more disulfide bonds. Purified heavy and light chains are by themselves virtually nontoxic, but when they are reassociated, toxicity is restored.Papain digesion of tetanus toxin results in cleavage of the heavy chain to give two fragments, B and C (8) (Fig. 1). Purified fragment C is completely nontoxic in animals, whereas fragment B retains some residual toxicity at high doses, although this activity is manifest as a flaccid paralysis in mice rather than the spastic paralysis characteristic of tetanus toxin (7,8).Immunity to tetanus toxin is provided by the administration of formaldehyde-treated toxin (tetanus toxoid). Fragments B and C have also been used to successfully immunize animals against tetanus, indicating that the entire molecule is not essential for protection (6). However, the preparation of these fragments is time-consuming and not commercially feasible. Studies with monoclonal antibodies have also shown that all three domains of the tetanus toxin molecule (the light chain, the amino-terminal half of the heavy chain, and the C fragment) may induce neutralizing antibodies (10, 25).To characterize tetanus toxin further at the molecular level, we undertook the cloning, nucleotide sequencing, and expression in Escherichia coli of DNA encoding fragment C of tetanus toxin. We used synthetic oligonucleotides com-* Corresponding author.plementary to the amino acid sequence of fragment C ...

Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis.

“…Several clones were identified that hybridized to the oligonucleotides, and one clone was studied further. To cohfirm that this cloned fragment did encode part of the P.69 gene, a Sac II subfragment of the 1.9-kb Pst I fragment was purified, BAL-31-treated, and ligated into the translatiof fusion vector pWRL507 (12). Several positive recombinants were obtained when the colonies were screened by colony blotting and immunoblotting with the mAb BB05 tdata not shown; see Fig.…”

“…The P.93 protein was expressed in E. coli as fusion proteins either with the trpE gene product by using the vector pWRL507 (12) or with the a peptide of B-galactosidase by using the pUC vectors (11). Plasmid pWDP311 (see above) expresses an 82-kDa protein (Fig.…”

“…The B. pertussis strains BP368, BP369, BP536, BP537, and BP571 were obtained from J. Miller (Stanford University). Escherichia coli strains TGl and HB101, cosmid pHC79, and plasmids pUC8, pUC9, and pWRL507 have been described (9)(10)(11)(12).…”

“…The protein sequence was determined by using an Applied Biosystems 470-A gas-phase microsequencer. Expression and detection of fragments of P.69 protein cloned in E. coli plasmids were as described (12).…”

Activation of Tumor-specific CD4+ T Lymphocytes by Major Histocompatibility Complex Class II Tumor Cell Vaccines

“…For the pLPCX/TT construct, TT fragment C DNA was PCR amplified from pCR Blunt (19) to include an ATG start codon and HindIII site at the 5Ј end and a BamHI site at the 3Ј end: 5Ј primer sequence, CCGCCGAAGCT-TGCCACCATGAAAAACCTTGATTGTT; and 3Ј primer sequence, CTGT-TCGGATCCTTAGTCGTTGGTCCAA. PCR conditions were: denature at 94°C for 5 min, denature at 94°C for 1 min, anneal at 55°C for 1 min, extend at 72°C for 1 min (TaqDNA polymerase; Invitrogen); repeat the last three steps 35 times and extend at 72°C for 10 min.…”