Enrichment for 5'-TG termini: a method for subcloning structural genes into expression vectors.
We describe a method for creating a population of randomly digested, blunt-ended DNA fragments with 5'-TG... 3'-AC... (TG) at their termini. When these fragments were ligated to a blunt-ended vector that contains ...CATA-3' ...GTAT-5' at its termini, as high as 84% of the clones obtained after transformation contained plasmids with a reconstructed Nde I site ... CATATG... ...GTATAC.... When the DNA vector is prepared from an appropriate plasmid [Gross et al., Mol. Cell. Biol. 5 (1985) 1015-1024; Kotewicz et al., Gene 35 (1985) 249-258], the ATG within the restriction site corresponds to a start codon positioned downstream from a strong ribosome-binding site and controllable promoter. If a gene has been digested to the TG contained within its authentic initiation codon, the endogenous translation-initiation control sequences are deleted and expression can be controlled using the plasmid-derived promoter. In addition, a gene digested to other in-frame TGs can potentially express proteins with altered N termini. Using this method, we have placed the structural gene of SP6 RNA polymerase, trimmed precisely to its authentic start codon, under the control of the tac promoter.
D'Alessio
J M
JM
Bethesda Research Laboratories, Life Technologies, Inc., Gaithersburg, MD 20877.
Hammond
A W
AW
Chatterjee
D K
DK
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon / Q000235:genetics; D004274:DNA, Recombinant; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005822:Genetic Vectors; D010957:Plasmids; D011401:Promoter Regions, Genetic
D003062:Codon; D004274:DNA, Recombinant; D012321:DNA-Directed RNA Polymerases