Expression of tetanus toxin fragment C in yeast: gene synthesis is required to eliminate fortuitous polyadenylation sites in AT-rich DNA.
Fragment C is a non-toxic 50 kDa fragment of tetanus toxin which is a candidate subunit vaccine against tetanus. The AT-rich Clostridium tetani DNA encoding fragment C could not be expressed in Saccharomyces cerevisiae due to the presence of several fortuitous polyadenylation sites which gave rise to truncated mRNAs. The polyadenylation sites were eliminated by chemically synthesising the DNA with increased GC-content (from 29% to 47%). Synthesis of the entire gene (1400 base pairs) was necessary to generate full-length transcripts and for protein production in yeast. Using a GAL1 promoter vector, fragment C was expressed to 2-3% of soluble cell protein. Fragment C could also be secreted using the alpha-factor leader peptide as a secretion signal. The protein was present at 5-10 mg/l in the culture medium in two forms: a high molecular mass hyper-glycosylated protein (75-200 kDa) and a core-glycosylated protein (65 kDa). Intracellular fragment C was as effective in vaccinating mice against tetanus authentic fragment C. The glycosylated material was inactive, though it was rendered fully active by de-glycosylation.
Romanos
M A
MA
Department of Molecular Biology, Wellcome Biotech, Beckenham, Kent, UK.
Makoff
A J
AJ
Fairweather
N F
NF
Beesley
K M
KM
Slater
D E
DE
Rayment
F B
FB
Payne
M M
MM
Clare
J J
JJ
D000225:Adenine / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D004271:DNA, Fungal / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005800:Genes, Fungal; D006031:Glycosylation; D007114:Immunization; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D010957:Plasmids; D011061:Poly A / Q000378:metabolism*; D012331:RNA, Fungal / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013742:Tetanus / Q000517:prevention & control; D013744:Tetanus Toxin / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D013936:Thymidine / Q000032:analysis; D014158:Transcription, Genetic
D004271:DNA, Fungal; D010446:Peptide Fragments; D012331:RNA, Fungal; D011994:Recombinant Proteins; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; D011061:Poly A; D000225:Adenine; D013936:Thymidine