Construction and use of lambda PL promoter vectors for direct cloning and high level expression of PCR amplified DNA coding sequences.
A set of plasmid vectors which allow single-step cloning and expression of PCR-amplified DNA coding sequences has been constructed. The vectors contain the phage lambda PL promoter, a synthetic translation initiation region (TIR), and convenient cloning sites. The cloning sites provide all or part of an AUG translation initiation codon and facilitate the precise fusion of target DNA sequences to vector transcriptional and translational signals. The vectors were constructed with synthetic TIRs because there is evidence which suggests that the efficiency of the phage lambda cII gene TIR present in the parental vector depends strongly on information contained within the cII N-terminal coding sequence. Bovine brain 14-3-3 eta chain cDNA was PCR-amplified and used to demonstrate the expression capacity of the newly constructed vectors. A significant increase in expression of 14-3-3 protein was observed when synthetic TIRs were used in the place of the cII TIR. Expression levels vary from 15% to 48% of total cell protein. The effects of a reported translational enhancer from phage T7 on expression of the 14-3-3 protein are also discussed. The vectors should be generally useful for high level heterologous protein expression in Escherichia coli.
Cheng
X
X
DuPont Merck Pharmaceutical Company, Wilmington, DE 19880-0400.
Patterson
T A
TA
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014764:Viral Proteins
D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; D014764:Viral Proteins; C039000:cII protein, bacteriophage lambda