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A New Strategy for Optimizing the Translation Initiation Rate of Foreign Gene Expression in E. coli.
MedLine Citation:
PMID:  12167985     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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 E. coli. As a model, the 5' 114 bp coding sequence (38 amino acids from the start codon) of human proliferating cell nuclear antigen (PCNA) 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 nucleotides 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' PCNA-LacZ' mRNA could be efficiently and specifically transcribed by inducible T7 RNA polymerase in E. coli strain JM 109 (DE3). There were 269 clones of 5' PCNA-LacZ' fusion plasmid selected first by the blue color on X-gal plate and then by hybridization with a 5' PCNA 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 human PCNA gene in E. coli was obtained.
Authors:
Yi-Lun Wang; Ying-Jun Gou; Min-Yan Qiu; Chang-De Lu
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Publication Detail:
Type:  JOURNAL ARTICLE    
Journal Detail:
Title:  Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica     Volume:  30     ISSN:  0582-9879     ISO Abbreviation:  Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao     Publication Date:  1998  
Date Detail:
Created Date:  2002-Aug-8     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  20730160R     Medline TA:  Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai)     Country:  -    
Other Details:
Languages:  ENG     Pagination:  544-549     Citation Subset:  -    
Affiliation:
Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China. lucd@sunm.shcnc.ac.cn
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