Expression of tetanus toxin fragment C in E. coli: high level expression by removing rare codons.
Tetanus toxin fragment C had been previously expressed in Escherichia coli at 3-4% cell protein. The codon bias for tetanus toxin in Clostridium tetani is very different from that of highly expressed homologous genes in E. coli, resulting in the presence of many rare E. coli codons in the sequence encoding fragment C. We have replaced the coding sequence by sequence optimized for codon usage in E. coli, and show that the expression of fragment C is increased. Although the level of mRNA also increased this appeared to be a secondary consequence of more efficient translation. Complete sequence replacement increased expression to approximately 11-14% cell protein but only after the promoter strength had been improved.
Makoff
A J
AJ
Department of Molecular Biology, Wellcome Biotech, Beckenham, Kent, UK.
Oxer
M D
MD
Romanos
M A
MA
Fairweather
N F
NF
Ballantine
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010446:Peptide Fragments / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D013744:Tetanus Toxin / Q000235:genetics*; D014158:Transcription, Genetic
D003062:Codon; D010446:Peptide Fragments; D012333:RNA, Messenger; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C