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  • Title: Expression of normal and transforming H-ras genes in Escherichia coli and purification of their encoded p21 proteins.
    Author: Lacal JC, Santos E, Notario V, Barbacid M, Yamazaki S, Kung H, Seamans C, McAndrew S, Crowl R.
    Journal: Proc Natl Acad Sci U S A; 1984 Sep; 81(17):5305-9. PubMed ID: 6089191.
    Abstract:
    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.
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