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Enhanced polypeptide synthesis programmed by linear DNA fragments in cell‐free extracts lacking exonuclease V
Author(s) -
Jackson Maria,
Pratt Julie M.,
Holland I.Barry
Publication year - 1983
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(83)80823-0
Subject(s) - exonuclease , dna , chemistry , microbiology and biotechnology , exonuclease iii , biochemistry , biology , gene , dna polymerase , escherichia coli
Preparation of an in vitro coupled transcription‐translation system from E. coli strains lacking exonuclease V has greatly improved the system for use with added linear DNA fragments. In fact, in extracts of these mutants linear fragments are stable for several hours. However the cell extracts show a high level of endogenous background. To avoid this complication extracts were prepared at 30°C from a mutant carrying a temperature‐sensitive exonuclease V. Polypeptides coded by a specific DNA region, e.g., delineated by restriction endonuclease sites, can now be easily identified.