Method for protein expression starting from stabilized linear short DNA in cell-free in vitro transcription/translation systems with exonuclease-containing lysates or in a cellular system containing exonucleases

摘要

The present invention concerns a method for protein expression comprising the steps of transcribing stabilized linear short DNA in cell-free in vitro transcription/ translation systems with exonuclease-containing lysates or in a cellular system containing exonucleases and subsequent translation, wherein the stability of the linear short DNA is improved by one or several of the following measures to protect the double-stranded DNA from exonucleases:a) incorporation of exonuclease resistant nucleotide analogues or other molecules at the 3' end of the template,b) use of PCR primer pairs which contain exonuclease-resistant nucleotides to produce a linear short DNA,c) protection of a template produced by a PCR reaction by connecting the 5' primer to the 3' end of the complementary strand,d) protecting the template by DNA sequence-specific binding molecules which bind to both ends of the linear template,e) inactivation of the exonucleases by adding competitive or non-competitive inhibitors,f) circularization of the template to form a ring-shaped closed template.