Expression of normal and transforming H-ras genes in Escherichia coli and purification of their encoded p21 proteins.
The H-ras gene of the BALB murine sarcoma virus (BALB-MSV) was placed under the transcriptional control of the tightly regulated PL promoter of bacteriophage lambda in the expression vectors pEV-vrf-1 and pRC23. Upon derepression of the PL promoter, large amounts (10-20% of total cellular protein) of the H-ras gene product p21 are synthesized in Escherichia coli. We constructed three H-ras gene expression vectors, designated pJCL-H5, pJCL-E30, and pJCL-33. pJCL-H5 directs the synthesis of p21, a fusion protein whose four amino-terminal residues are replaced by eight amino acids coded for by plasmid sequences. The 13 5' coding nucleotides of the BALB-MSV H-ras gene missing in pJCL-H5 were regenerated in pJCL-E30 by inserting a pair of complementary synthetic oligodeoxynucleotides. As a result, pJCL-E30 encodes a p21 protein, p21T, of sequence identical to that of the transforming p21 protein of BALB-MSV. pJCL-33 is a derivative of pJCL-E30 in which the 12th codon, AAA, a lysine codon, was replaced by GGA, a glycine codon. Thus, pJCL-33 directs the synthesis of a p21 protein, p21N, whose sequence corresponds to that of a normal cellular p21 protein. We report the purification of H-ras p21 proteins to apparent homogeneity by a method involving solubilization with chaotropic agents followed by reverse-phase high-performance liquid chromatography.
D000818:Animals; D001483:Base Sequence; D002471:Cell Transformation, Neoplastic; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005814:Genes, Viral; D005822:Genetic Vectors; D051379:Mice; D008807:Mice, Inbred BALB C; D009363:Neoplasm Proteins / Q000235:genetics* / Q000302:isolation & purification; D009857:Oncogenes; D009876:Operon; D010957:Plasmids; D016283:Proto-Oncogene Proteins p21(ras); D009053:Sarcoma Viruses, Murine / Q000235:genetics*; D014158:Transcription, Genetic
D009363:Neoplasm Proteins; D004262:DNA Restriction Enzymes; D016283:Proto-Oncogene Proteins p21(ras)