A New Strategy for Optimizing the Translation Initiation Rate of Foreign Gene Expression in XXXX .

@entity1 @entity1522 @entity1364 @entity2036 @entity526
The translation initiation rate is greatly affected by the secondary structure of the translation initiation region (TIR) of mRNA. A novel system was established for improving the translation initiation rate of a foreign gene in @entity526 . As a model, the 5' 114 bp coding sequence (38 @entity1522 from the start codon) of @entity1 @entity1364 ( @entity1364 ) gene was fused with the lacZ' gene at its 5' end in vector pTZ19R. A Shine/Dalgarno (SD) sequence GAGGT was inserted to the -8 position of AUG by site-directed mutation. Then the flanking sequences of SD, which were the 6 @entity2036 upstream the SD and the 7 nucleotides between SD and AUG, were randomly changed by PCR using a synthetic primer with partially random sequences. This random mutation led to potential variations in the secondary structure of the TIR of mRNA through base pairing with the 5' coding sequence. The 5' @entity1364 -LacZ' mRNA could be efficiently and specifically transcribed by inducible T7 RNA polymerase in @entity526 JM 109 (DE3). There were 269 clones of 5' @entity1364 -LacZ' fusion plasmid selected first by the blue color on X-gal plate and then by hybridization with a 5' @entity1364 probe. Eight clones with different blue colors among the 269 clones were chosen for beta-galactosidase activity assay. The results showed that the difference between their enzyme activities was more than 20 fold, but there seemed no apparent difference at the transcription level as assayed by RNA dot hybridization, which suggested that the difference in the expression of the fusion protein was due to the different rate of translation initiation. Thus, by this strategy, an effective translation initiation region for the high expression of @entity1 @entity1364 gene in @entity526 was obtained.
