title: High-level expression of tetanus toxin fragment C-thioredoxin fusion protein in Escherichia coli.
abstract: An insert of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by PCR. This 1.4 kb fragment was cloned into the high-expression vector pET32a, under control of the T7 promoter. Expression of this plasmid in Escherichia coli BL21(DE3) resulted in the production of a fusion protein ( approximately 62 kDa) consisting of 112 amino acids of thioredoxin and approximately 450 amino acids of fragment C. This fusion protein was recognized by anti-tetanus toxoid antiserum in an ELISA and on immunoblots. The recombinant fragment-C-thioredoxin protein was purified significantly in one step by Ni(2+)-chelate Sepharose, the final yield being approximately 35 mg/l. Immunization of animals with the recombinant protein produced antibodies that were able to recognize the tetanus toxin. By using this gene-fusion expression system we produced soluble fragment C of tetanus toxin in a high yield, preventing many problems inherent in the use of other expression systems that produce either insoluble fragment C in inclusion bodies, or a soluble form, but in low yield, using E. coli as the expression host.
authors.affiliation: Center of Biotechnology, Instituto Butantan, Av. Vital Brasil, 1500, CEP 05503-900, São Paulo, SP, Brazil.
authors.forename: A V
authors.initials: AV
authors.lastname: Ribas
authors.forename: P L
authors.initials: PL
authors.lastname: Ho
authors.forename: M M
authors.initials: MM
authors.lastname: Tanizaki
authors.forename: I
authors.initials: I
authors.lastname: Raw
authors.forename: A L
authors.initials: AL
authors.lastname: Nascimento
mesh_terms: D001483:Base Sequence; D001709:Biotechnology; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013744:Tetanus Toxin / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D013879:Thioredoxins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; D013879:Thioredoxins