Production of chimeric protein coded by the fused viral H-ras and human N-ras genes in Escherichia coli.
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 lambda 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.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002678:Chimera; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005814:Genes, Viral; D006801:Humans; D009857:Oncogenes; D010957:Plasmids; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras)
D011518:Proto-Oncogene Proteins; D004262:DNA Restriction Enzymes; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)