The translation initiation site of recombinant Trypanosoma brucei ornithine decarboxylase varies with different promoters.
Expression of the Trypanosoma brucei ornithine decarboxylase (ODC) gene in Escherichia coli behind the lambda phage PR promoter led to the production of a recombinant enzyme having the same subunit molecular weight as the native enzyme [4]. However, when the same gene is expressed behind the tac promoter or the phoA promoter, the ODCs produced by the transformed E. coli have subunit molecular weights approximately 2 kDa higher than that of the native enzyme. Amino terminal sequencing of the recombinant proteins indicates that the ODC synthesized under control of the lambda PR promoter actually starts at the second methionine (Met23) of the open reading frame, whereas those produced in the latter two cases begin at the first methionine (Met1). Analysis of the 5'-end of T. brucei ODC mRNA supports the conclusion that translation initiates at Met23. We postulate that, for the lambda PR promoter, translation initiates at Met23 instead of Met1 because of the formation of a stable secondary structure in the region of the Met1 and the presence of a good E. coli consensus translation initiation site upstream of Met23. We have constructed a new plasmid using the pho A promoter to express recombinant T. brucei ODC starting at Met23 in large quantities.
Kuntz
D A
DA
Department of Pharmaceutical Chemistry, University of California, San Francisco.
Phillips
M A
MA
Moore
T D
TD
Craig
S P
SP
Bass
K E
KE
Wang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015342:DNA Probes; D016054:DNA, Protozoan / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009955:Ornithine Decarboxylase / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry; D016053:RNA, Protozoan / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics*
D015342:DNA Probes; D016054:DNA, Protozoan; D012333:RNA, Messenger; D016053:RNA, Protozoan; D011994:Recombinant Proteins; D009955:Ornithine Decarboxylase