title: Expression of fragment C of tetanus toxin fused to a carboxyl-terminal fragment of diphtheria toxin in Salmonella typhi CVD 908 vaccine strain.
abstract: We report the expression of fragment C of tetanus toxin (FC) fused to the eukaryotic cell binding domain (the carboxyl-terminus) of diphtheria toxin (FC-bDt fusion) in attenuated Salmonella typhi live vector vaccine strain CVD 908. The FC-bDt protein fusion was constructed using plasmid pTETnir15 which carries the gene encoding FC under control of the nirB promoter (nirBP). The open reading frame for FC was modified to incorporate an in-frame glycine-proline hinge region and a set of four restriction sites at the 3' end of the FC gene. A 482 bp DNA fragment encoding the eukaryotic cell binding domain of diphtheria toxin was then inserted at the 3' end of the modified FC gene to create an in-frame FC-bDt fusion gene. The resulting plasmid, pOG215, was able to express the FC-bDt fusion protein in both Escherichia coli DH5a and S. typhi CVD 908, as evidenced by Western immunoblots using anti-FC and anti-C-terminal diphtheria toxin monoclonal antibodies. Maximum expression of the FC-bDt fusion protein was achieved by growing CVD 908(pOG215) at the low oxidation-reduction potential of thioglycollate broth, i.e. in conditions that activate nirBP and drive transcription of the FC-bDt fusion gene. Whereas maximum expression of FC alone was also observed using thioglycollate broth, expression of bDt alone was unsuccessful using a variety of growth conditions. FC fusions constitute one strategy to "rescue" expression of proteins which are otherwise difficult to express.
authors.affiliation: Department of Medicine, University of Maryland School of Medicine, Baltimore 21201, USA.
authors.forename: O G
authors.initials: OG
authors.lastname: Gómez-Duarte
authors.forename: J
authors.initials: J
authors.lastname: Galen
authors.forename: S N
authors.initials: SN
authors.lastname: Chatfield
authors.forename: R
authors.initials: R
authors.lastname: Rappuoli
authors.forename: L
authors.initials: L
authors.lastname: Eidels
authors.forename: M M
authors.initials: MM
authors.lastname: Levine
mesh_terms: D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics; D001428:Bacterial Vaccines / Q000235:genetics; D001483:Base Sequence; D004167:Diphtheria Toxin / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012485:Salmonella typhi / Q000235:genetics / Q000378:metabolism*; D013744:Tetanus Toxin / Q000096:biosynthesis* / Q000235:genetics; D014613:Vaccines, Attenuated / Q000235:genetics
chemical_list: D000942:Antigens, Bacterial; D001428:Bacterial Vaccines; D004167:Diphtheria Toxin; D010446:Peptide Fragments; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; D014613:Vaccines, Attenuated