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Production of chimeric protein coded by the fused viral H-rasand human N-rasgenes inEscherichia coli

Authors
Journal
Gene
0378-1119
Publisher
Elsevier
Publication Date
Volume
52
Identifiers
DOI: 10.1016/0378-1119(87)90048-5
Keywords
  • Recombinant Dna
  • Autophosphorylation
  • λ Exonuclease
  • Expression Plasmid
  • Inclusion Bodies
Disciplines
  • Biology

Abstract

Abstract A new method for the production of a chimeric protein of two related genes has been developed. The nucleotide sequences of the region from the N terminus to the 86th amino acid (aa) residue of human N-ras and of the Harvey sarcoma virus (Ha-MuSV) H- ras are 80% homologous. We isolated the DNA fragment encoding the N-terminal portion up to the 70th aa residue from plasmid pH-1 which encodes the total genome of Ha-MuSV, and the DNA fragment encoding the C-terminal portion from the 40th aa to the C terminus from plasmid p6a1 which includes the human N- ras cDNA but lacks the N-terminal portion. After partial digestion of both fragments with phage λ exonuclease, which creates 3′-protruding ends, a hybrid was formed between 73% homologous single-stranded DNA portions at the 3′ ends of both fragments. The hybrid was recloned on pBR322 after repairing with Escherichia coli DNA polymerase I and DNA ligase. The chimeric v-H/N- ras gene composed of the N-terminal portion of v-H- ras gene and the remaining region of N- ras gene was inserted into an expression vector containing two tandem trp promoters and a terminator, and expressed in E. coli. The chimeric protein was found to accumulate to approx. 10% of total cellular proteins.

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